Modified rsv f proteins and methods of their use

ABSTRACT

The present invention is generally related to modified or mutated respiratory syncytial virus fusion (F) proteins and methods for making and using them, including immunogenic compositions such as vaccines for the treatment and; or prevention of RSV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/599,568, filedOct. 11, 2019, which is a continuation of U.S. Ser. No. 16/009,257,filed. Jun. 15, 2018, now U.S. Pat. No. 11,052,146, issued Jul. 6, 2021,which is a continuation of U.S. Ser. No. 14/839,247, filed Aug. 28,2015, now U.S. Pat. No. 10,022,437, issued Jul. 17, 2018, which is acontinuation of U.S. Ser. No, 14/221,675, filed Mar. 21, 2014, now U.S.Pat. No. 9,675,685, issued Jun. 13, 2017, which is a continuation ofU.S. Ser. No. 12/633,995, filed Dec. 9, 2009, now U.S. Pat. No.8,715,692, issued May 6, 2014, which claims priority to U.S. ProvisionalApplication Ser. No. 61/121,126, filed Dec. 9, 2008, U.S. ProvisionalApplication Ser. No. 61/169,077, filed Apr. 14, 2009, and U.S.Provisional Application Ser. No. 61/224,787, filed Jul. 10, 2009, eachof which is herein incorporated by reference in its entirety for allpurposes.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(NOVV_034_09US_SeqList_ST26.xml; Size: 62,550 bytes; and 17ate ofCreation: Aug. 4, 2022) is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention is generally related to modified or mutatedrespiratory syncytial virus fusion (F) proteins and methods for makingand using them, including immunogenic compositions such as vaccines forthe treatment and/or prevention of RSV infection.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is a member of the genus Pneumovirusof the family Paromyxaviridae. Human RSV (HRSV) is the leading cause ofsevere lower respiratory tract disease in young children and isresponsible for considerable morbidity and mortality in humans. RSV isalso recognized as an important agent of disease in immunocompromisedadults and in the elderly. Due to incomplete resistance to RSV in theinfected host after a natural infection, RSV may infect multiple timesduring childhood and adult life.

This virus has a genome comprised of a single strand negative-sense RNA,which is tightly associated with viral protein to form the nucleocapsid.The viral envelope is composed of a plasma membrane derived lipidbilayer that contains virally encoded structural proteins. A viralpolymerase is packaged with the virion and transcribes genomic RNA intomRNA, The RSV genome encodes three transmembrane structural proteins, F,G, and SH, two matrix proteins, M and M2, three nucleocapsid proteins N,P, and L, and two nonstructural proteins, NS1 and NS2.

Fusion of HRSV and cell membranes is thought to occur at the cellsurface and is a necessary step for the transfer of viralribonucleoprotein into the cell cytoplasm during the early stages ofinfection. This process is mediated by the fusion (F) protein, whichalso promotes fusion of the membrane of infected cells with that ofadjacent cells to form a characteristic syncytia, which is both aprominent cytopathic effect and an additional mechanism of viral spread.Accordingly, neutralization of fusion activity is important in hostimmunity. Indeed, monoclonal antibodies developed against the F proteinhave been shown to neutralize virus infectivity and inhibit membranefusion (Calder et al., 2000, Virology 271: 122-131).

The F protein of RSV shares structural features and limited, butsignificant amino acid sequence identity with F glycoproteins of otherparamyxoviruses. It is synthesized as an inactive precursor of 574 aminoacids (F0) that is cotranslationally glycosylated on asparagines in theendoplasmic reticulum, where it assembles into homo-oligomers. Beforereaching the cell surface, the F0 precursor is cleaved by a proteaseinto F2 from the N terminus and F1 from the C terminus. The F2 and F1chains remains covalently linked by one or more disulfide bonds.

Immunoaffinity purified full-length F proteins have been found toaccumulate in the form of micelles (also characterized as rosettes),similar to those observed with other full-length virus membraneglycoproteins (Wrigley et 1986, in Electron Microscopy of Proteins, Vol5, p. 103-163, Academic Press, London). Under electron microscopy, themolecules in the rosettes appear either as inverted cone-shaped rods(˜70%) or lollipop-shaped (˜30%) structures with their wider endsprojecting away from the centers of the rosettes. The rod conformationalstate is associated with an F glycoprotein in the pre-fusion inactivatestate while the lollipop conformational state is associated with an Fglycoprotein in the post-fusion, active state.

Electron micrography can be used to distinguish between the prelusionand postfusion (alternatively designated prefusogenic and fusogenic)conformations, as demonstrated by Calder et al., 2000. Virology271:122-131. The prefusion conformation can also be distinguished fromthe fusogenic (postfusion) conformation by liposome association assays.Additionally, prefusion and fusogenic conformations can be distinguishedusing antibodies (e.g., monoclonal antibodies) that specificallyrecognize conformation epitopes present on one or the other of theprefusion or fusogenic form of the RSV F protein, but not on the otherform. Such conformation epitopes can be due to preferential exposure ofan antigenic determinant on the surface of the molecule. Alternatively,conformational epitopes can arise from the juxtaposition of amino acidsthat are non-contiguous in the linear polypeptide.

It has been shown previously that the F precursor is cleaved at twosites (site 1, after residue 109 and site 11, after residue 136), bothpreceded by motifs recognized by furin-like proteases. Site 11 isadjacent to a fusion peptide, and cleavage of the F protein at bothsites is needed for membrane fusion (Gonzalez-Reyes et al., 2001, PNAS98(17): 9859-9864). When cleavage is completed at both sites, it isbelieved that there is a transition from cone-shaped to lollipop-shapedrods.

SUMMARY OF THE INVENTION

As described herein, the present inventors have found that surprisinglyhigh levels of expression of the fusion (F) protein can be achieved whencertain modifications are made to the structure of the RSV F protein.Such modifications also unexpectedly reduce the cellular toxicity of theRSV F protein in a host cell. In addition, the modified F proteins ofthe present invention demonstrate an improved ability to exhibit thepost-fusion “lollipop” morphology as opposed to the pre-fusion “rod”morphology. Thus, in one aspect, the modified F proteins of the presentinvention can also exhibit improved immunogenicity as compared towild-type F proteins. These modifications have significant applicationsto the development of vaccines and methods of using said vaccines forthe treatment and/or prevention of RSV. The present invention providesrecombinant RSV F proteins that demonstrate increased expression,reduced cellular toxicity, and/or enhanced immunogenic properties ascompared to wild-type RSV F proteins.

In one aspect, the invention provides recombinant RSV F proteinscomprising modified or mutated amino acid sequences as compared towild-type RSV F proteins. In general, these modifications or mutationsincrease the expression, reduce the cellular toxicity, and/or enhancethe immunogenic properties of the RSV F proteins as compared towild-type RSV F proteins. In certain exemplary embodiments, the RSV Fproteins are human RSV F proteins.

The RSV F protein preferably comprises a modified or mutated amino acidsequence as compared to the wild-type RSV F protein (e.g. as exemplifiedin SEQ ID NO: 2). In one embodiment, the RSV F protein contains amodification or mutation at the amino acid corresponding to positionP102 of the wild-type RSV F protein (SEQ ID NO: 2). In anotherembodiment, the RSV F protein contains a modification or mutation at theamino acid corresponding to position I379 of the wild-type RSV F protein(SEQ ID NO: 2). In another embodiment, the RSV F protein contains amodification or mutation at the amino acid corresponding to positionM447 of the wild-type RSV F protein (SEQ ID NO: 2).

In one embodiment, the RSV F protein contains two or more modificationsor mutations at the amino acids corresponding to the positions describedabove. In another embodiment, the RSV F protein contains threemodifications or mutations at the amino acids corresponding to thepositions described above.

In one specific embodiment, the invention is directed to RSV F proteinswherein the proline at position 102 is replaced with alanine. In anotherspecific embodiment, the invention is directed to RSV F proteins whereinthe isoleucine at position 379 is replaced with valine. In yet anotherspecific embodiment, the invention is directed to RSV F proteins whereinthe methionine at position 447 is replaced with valine. In certainembodiments, the RSV F protein contains two or more modifications ormutations at the amino acids corresponding to the positions described inthese specific embodiments. In certain other embodiments, the RSV Fprotein contains three modifications or mutations at the amino acidscorresponding to the positions described in these specific embodiments.In an exemplary embodiment, the RSV protein has the amino acid sequencedescribed in SEQ ID NO: 4.

In one embodiment, the coding sequence of the RSV F protein is furtheroptimized to enhance its expression in a suitable host cell. In oneembodiment, the host cell is an insect cell. In an exemplary embodiment,the insect cell is an Sf9 cell.

In one embodiment, the coding sequence of the codon optimized RSV F geneis SEQ ID NO: 3. In another embodiment, the codon optimized RSV Fprotein has the amino acid sequence described in SEQ ID NO: 4.

In one embodiment, the RSV F protein further comprises at least onemodification in the cryptic poly(A) site of F2. In another embodiment,the RSV F protein further comprises one or more amino acid mutations atthe primary cleavage site (CS). In one embodiment, the RSV F proteincontains a modification or mutation at the amino acid corresponding toposition R133 of the wild-type RSV F protein (SEQ ID NO: 2) or the codonoptimized RSV F protein (SEQ NO: 4), In another embodiment, the RSV Fprotein contains a modification or imitation at the amino acidcorresponding to position R135 of the wild-type RSV F protein (SEQ IDNO: 2) or the codon optimized RSV F protein (SEQ ID NO: 4). In yetanother embodiment, the RSV F protein contains a modification ormutation at the amino acid corresponding to position 8136 of thewild-type RSV F protein (SEQ 11) NO: 2) or the codon optimized RSV Fprotein (SEQ ID NO: 4).

In one specific embodiment, the invention is directed to RSV F proteinswherein the arginine at position 133 is replaced with glutamine. Inanother specific embodiment, the invention is directed to RSV F proteinswherein the arginine at position 135 is replaced with glutamine. In yetanother specific embodiment, the invention is directed to RSV F proteinswherein arginine at position 136 is replaced with glutamine. In certainembodiments, the RSV F protein contains two or more modifications ormutations at the amino acids corresponding to the positions described inthese specific embodiments. In certain other embodiment, the RSV Fprotein contains three modifications or mutations at the amino acidscorresponding to the positions described in these specific embodiments.In an exemplary embodiment, the RSV protein has the amino acid sequencedescribed in SEQ ID NO: 6.

In another embodiment, the RSV F protein further comprises a deletion inthe N-terminal half of the fusion domain corresponding to amino acids137-146 of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6. In an exemplaryembodiment, the RSV F protein has the amino acid sequence described inSEQ ID NO: 8. In an alternative embodiment, the RSV F protein has theamino acid sequence described in SEQ 11) NO: 10.

Further included within the scope of the invention are RSV F proteins,other than human RSV F protein (SEQ ID NO: 2), which contain alterationscorresponding to those set out above. Such RSV F proteins may include,but are not limited to, the RSV F proteins from A strains of human RSV,B strains of human RSV, strains of bovine RSV, and strains of avian RSV.

In some embodiments, the invention is directed to modified or mutatedRSV F proteins that demonstrate increased expression in a host cell ascompared to wild-type RSV F proteins, such as the one shown by SEQ IDNO: 2. In other embodiments, the invention is directed to modified ormutated RSV F proteins that demonstrate reduced cellular toxicity in ahost cell as compared to wild-type RSV F proteins, such as the one shownby SEQ ID NO: 2. In yet other embodiments, the invention is directed tomodified or mutated RSV F proteins that demonstrate enhanced immunogenicproperties as compared to wild-type RSV F proteins, such as the oneshown by SEQ ID NO: 2.

In additional aspects, the invention provides immunogenic compositionscomprising one or more modified or mutated RSV F proteins as describedherein. In one embodiment, the invention provides a micelle comprised ofone or more modified or mutated RSV F proteins (e.g. an RSV F micelle).

In another embodiment, the present invention provides a virus-likeparticle (VLP) comprising a modified or mutated RSV F protein. In someembodiments, the VLP further comprises one or more additional proteins.

In one embodiment, the VLP further comprises a matrix (M) protein. Inone embodiment, the M protein is derived from a human strain of RSV. Inanother embodiment, the M protein is derived from a bovine strain ofRSV. In other embodiments, the matrix protein may be an M1 protein froman influenza virus strain. In one embodiment, the influenza virus strainis an avian influenza virus strain. In other embodiments, the M proteinmay be derived from a Newcastle Disease Virus (NDV) strain.

In additional embodiments, the VLP further comprises the RSVglycoprotein G. In another embodiment, the VLP further comprises the RSVglycoprotein SH. In yet another embodiment, the VLP further comprisesthe RSV nucleocapsid N protein.

The modified or mutated RSV F proteins may be used for the preventionand/or treatment of RSV infection. Thus, in another aspect, theinvention provides a method for eliciting an immune response againstRSV. The method involves administering an immunologically effectiveamount of a composition containing a modified or mutated RSV F proteinto a subject, such as a human or animal subject.

In another aspect, the present invention provides pharmaceuticallyacceptable vaccine compositions comprising a modified or mutated RSV Fprotein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein.

In one embodiment, the invention comprises an immunogenic formulationcomprising at least one effective dose of a modified or mutated RSV Fprotein. In another embodiment, the invention comprises an immunogenicformulation comprising at least one effective dose of an RSV F micellecomprising a modified or mutated RSV F protein. In yet anotherembodiment, the invention comprises an immunogenic formulationcomprising at least one effective dose of a VLP comprising a modified ormutated RSV F protein.

In another embodiment, the invention provides for a pharmaceutical packor kit comprising one or more containers filled with one or more of theingredients of the vaccine formulations of the invention.

In another embodiment, the invention provides a method of formulating avaccine or antigenic composition that induces immunity to an infectionor at least one disease symptom thereof to a mammal, comprising addingto the formulation an effective dose of a modified or mutated RSV Fprotein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein. In apreferred embodiment, the infection is an RSV infection.

The modified or mutated RSV F proteins of the invention are useful forpreparing compositions that stimulate an immune response that confersimmunity or substantial immunity to infectious agents. Thus, in oneembodiment, the invention provides a method of inducing immunity toinfections or at least one disease symptom thereof in a subject,comprising administering at least one effective dose of a modified ormutated RSV F protein, an RSV F micelle comprising a modified or mutatedRSV F protein, or a VLP comprising a modified or mutated RSV F protein.

In yet another aspect, the invention provides a method of inducingsubstantial immunity to RSV virus infection or at least one diseasesymptom in a subject, comprising administering at least one effectivedose of a modified or mutated RSV F protein, an RSV F micelle comprisinga modified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein.

Compositions of the invention can induce substantial immunity in avertebrate (e.g. a human) when administered to the vertebrate. Thus, inone embodiment, the invention provides a method of inducing substantialimmunity to RSV virus infection or at least one disease symptom in asubject, comprising administering at least one effective dose of amodified or mutated RSV F protein, an RSV F micelle comprising amodified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein. In another embodiment, the invention provides amethod of vaccinating a mammal against RSV comprising administering tothe mammal a protection-inducing amount of a modified or mutated RSV Fprotein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein.

In another embodiment, the invention comprises a method of inducing aprotective antibody response to an infection or at least one symptomthereof in a subject, comprising administering at least one effectivedose of a modified or mutated RSV F protein, an RSV F micelle comprisinga modified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein.

In another embodiment, the invention comprises a method of inducing aprotective cellular response to RSV infection or at least one diseasesymptom in a subject, comprising administering at least one effectivedose of a modified or mutated RSV F protein. In another embodiment, theinvention comprises a method of inducing a protective cellular responseto RSV infection or at least one disease symptom in a subject,comprising administering at least one effective dose of an RSV F micellecomprising a modified or mutated. RSV F protein. In yet anotherembodiment, the invention comprises a method of inducing a protectivecellular response to RSV infection or at least one disease symptom in asubject, comprising administering at least one effective dose of a VLP,wherein the VLP comprises a modified or mutated RSV F protein.

In yet another aspect, the invention provides an isolated nucleic acidencoding a modified or mutated RSV F protein of the invention. In anexemplary embodiment, the isolated nucleic acid encoding a modified ormutated RSV F protein is selected from the group consisting of SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

In yet another aspect, the invention provides an isolated cellcomprising a nucleic acid encoding a modified or mutated RSV F proteinof the invention. In an exemplary embodiment, the isolated nucleic acidencoding a modified or mutated RSV F protein is selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

In yet another aspect, the invention provides a vector comprising anucleic acid encoding a modified or mutated RSV F protein of theinvention. In an exemplary embodiment, the isolated nucleic acidencoding a modified or mutated RSV F protein is selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.In one embodiment, the vector is a baculovirus vector.

In yet another aspect, the invention provides a method of making a RSV Fprotein, comprising (a) transforming a host cell to express a nucleicacid encoding a modified or mutated RSV F protein of the invention; and(b) culturing said host cell under conditions conducive to theproduction of said RSV F protein. In one embodiment, the nucleic acidencoding a modified or mutated RSV F protein is selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.In another embodiment, the host cell is an insect cell. In a furtherembodiment, the host cell is an is an insect cell transfected with abaculovirus vector comprising a modified or mutated RSV F protein of theinvention.

In yet another aspect, the invention provides a method of making a RSV Fprotein micelle, comprising (a) transforming a host cell to express anucleic acid encoding a modified or mutated RSV F protein of theinvention; and (b) culturing said host cell under conditions conduciveto the production of said RSV F protein micelle. In one embodiment, thenucleic acid encoding a modified or mutated RSV F protein is selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 5. SEQ ID NO: 7,or SEQ ID NO: 9. In one embodiment, the host cell is an insect cell. Inan exemplary embodiment, the host cell is an is an insect celltransfected with a baculovirus vector comprising a modified or mutatedRSV F protein of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts structure of wild type HRSV F₀ protein comprising thecleavage site regions NNRARRELP (SEQ ID NO:30) and LSKKRKRRFLG (SEQ IDNO:31).

FIG. 2 depicts structures of modified RSV F₀ proteins with cleavage sitemutations as described in Example 3. The cleavage site regions are shownas NNRARRELP (SEQ NO:30) and LSKKRKRRFLG (SEQ ID NO:31). Particularcleavage site sequences are shown as KKQKQQ (SEQ ID NO:28), GRRQQR (SEQID NO:29), KKQKRQ (SEQ ID NO:32), RAQQ (SEQ ID NO:33), and RANK (SEQ IDNO:34).

FIG. 3 depicts conservative substitutions (R133Q, R135Q and R136Q) inthe primary cleavage site of modified HRSV F protein BV #541 (SEQ ID NO:6). The mutated cleavage site region is shown as LSKKQKQQFLG (SEQ IDNO:35).

FIG. 4 depicts sequence and structure of modified. HRSV F protein BV#541 (SEQ ID NO: 6), and the sequence of the KKQKQQ cleavage site (SEQID NO:28).

FIG. 5 depicts sequence and structure of modified HRSV F protein BV #622(SEQ ID NO: 10), and the KKRKRR cleavage site (SEQ ID NO:24),

FIG. 6 depicts SDS-PAGE coomassie-stained gel of purified recombinantHRSV F protein BV #622 with or without the presence of βME.

FIG. 7 depicts structure of modified HRSV F protein BV #683, and theKKQKQQ cleavage site (SEQ ID NO: 28).

FIG. 8 depicts SDS-PAGE coomassie-stained gels of purified recombinantHRSV F proteins BV #622 and BV #683 with or without the presence of βME(on the left), and their structures; as well as the KKQKQQ cleavage site(SEQ ID NO:28) and the KKRKRRcleavage site (SEQ ID NO:24).

FIG. 9 depicts SDS-PAGE coomassie-stained gel (on the left) and WesternBlot (on the right) analysis of purified recombinant HRSV F protein BV#683 with or without the presence of βME.

FIG. 10 depicts SDS-PAGE coomassie-stained gel used in purity analysisby scanning densitometry (on the left) and Western Blot (on the right)of purified recombinant HRSV F protein BV #683.

FIG. 11 depicts images of purified recombinant HRSV F protein BV #683micelles (rosettes) taken in negative stain electron microscopy.

FIG. 12 depicts particle size analysis of HRSV F protein BY #683micelles.

FIG. 13 depicts SDS-PAGE coomassie-stained gel (on the left) andWestern. Blot (on the right) analysis of modified HRSV F proteins BV#622 and BV #623 (SEQ ID NO: 21) with or without co-expression with HRSVN and BRSV M proteins in the crude cell culture harvests (intracellular)or pelleted samples by 30% sucrose gradient separation, and structuresof BV #622 and BV #623. Also featured is the KKRKRRcleavage site (SEQ IDNO:24)

FIG. 14 depicts SDS-PAGE coomassie-stained gel (on the left) and WesternBlot (on the right) analysis of modified HRSV F protein BV #622, doubletandem chimeric BV #636 (BV #541+BRSV M), BV #683, BV #684 (BV #541 withYIAL L-domain), and BV #685 (BV #541 with YKKL L-domain) with or withoutco-expression with HRSV N and BRSV M proteins in the crude cell cultureharvests (intracellular) samples, and structure of each analyzedmodified HRSV F protein. Also featured is the KKQKQQ cleavage site (SEQID NO:28), and the KKRKRRcleavage site (SEQ ID NO:24).

FIG. 15 depicts SDS-PAGE coomassie-stained gel (on the left) and WesternBlot (on the right) analysis of modified RSV F protein BV #622 (SEQ IDNO: 10), double tandem chimeric BV #636 (BV #541 -BRSV M), BV #683 (SEQID NO: 8), BV #684 (BV #541 with YIAL L-domain), and BV #685 (BV #541with YKKL L-domain) with or without co-expression with HRSV N and BRSV Mproteins in the pelleted samples by 30% sucrose gradient separation, andstructure of each analyzed modified HRSV F protein. Also featured is theKKQKQQ cleavage site (SEQ ID NO:28), and the KKRKRR cleavage site (SEQID NO:24).

FIGS. 16A-16D depict structure, clone name, description, Western Blotand SDS-PAGE coomassie results, and conclusion for each modified RSV Fprotein as described in Example 9. Also featured are the KKQKQQ (SEQ IDNO:28), GRRQQR (SEQ ID NO:29), RANN (SEQ ID NO:34), RARR (SEQ NO:23),and KKRKRR (SEQ ID NO:24) cleavage site sequences.

FIG. 17 depicts experimental procedures of the RSV challenge study asdescribed in Example 10.

FIG. 18 depicts results of RSV neutralization assay at day 31 and day 46of mice immunized with PBS, live RSV, FI-RSV, 1 ug PFP. 1 ug PFP+Alum,10 ug PFP 10 ug PFP+Alum, 30 ug PEP, and positive control (anti-Fsheep).

FIG. 19 depicts RSV titers in lung tissues of mice immunized with PBSlive RSV, FI-RSV 1 ug PFP, 1 ug PFP+Alum, 10 ug PFP, 10 ug PFP+Alum and30 ug PFP, 4 days after challenge of infectious RSV.

FIG. 20 depicts SDS-PAGE gel stained with coomassie of purifiedrecombinant RSV F protein BV #683 stored at 2-8° C. for 0, 1, 2, 4, and5 weeks.

FIG. 21 depicts RSV A and RSV B neutralizing antibody responsesfollowing immunization with live RSV (RSV), formalin inactivated RSV(FI-RSV), RSV-F protein BV #683 with and without aluminum (PFP andPFP+Aluminum Adjuvant), and PBS controls.

FIG. 22 depicts lung pathology following challenge with RSV in ratsimmunized with live RSV, formalin inactivated RSV (FI-RSV), RSV-Fprotein BV #683 with and without aluminum (F-micelle (30 ug) andF-micelle (30 ug)+Aluminum Adjuvant), and PBS controls.

DETAILED DESCRIPTION Definitions

As used herein the term “adjuvant” refers to a compound that, when usedin combination with a specific immunogen (e.g. a modified or mutated RSVF protein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein) in aformulation, will augment or otherwise alter or modify the resultantimmune response. Modification of the immune response includesintensification or broadening the specificity of either or both antibodyand cellular immune responses. Modification of the immune response canalso mean decreasing or suppressing certain antigen-specific immuneresponses.

As use herein, the term “antigenic formulation” or “antigeniccomposition” refers to a preparation which, when administered to avertebrate, especially a bird or a mammal, will induce an immuneresponse.

As used herein the term “avian influenza virus” refers to influenzaviruses found chiefly in birds but that can also infect humans or otheranimals. In some instances, avian influenza viruses may be transmittedor spread from one human to another. An avian influenza virus thatinfects humans has the potential to cause an influenza pandemic, i.e.,morbidity and/or mortality in humans. A pandemic occurs when a newstrain of influenza virus (a virus in which human have no naturalimmunity, emerges, spreading beyond individual localities, possiblyaround the globe, and infecting many humans at once.

As used herein an “effective dose” generally refers to that amount of amodified or mutated RSV F protein, an RSV F micelle comprising amodified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein of the invention sufficient to induce immunity, toprevent and/or ameliorate an infection or to reduce at least one symptomof an infection or disease, and/or to enhance the efficacy of anotherdose of a modified or mutated RSV F protein, an RSV F micelle comprisinga modified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein. An effective dose may refer to the amount of amodified or mutated RSV F protein, an RSV F micelle comprising amodified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein sufficient to delay or minimize the onset of aninfection or disease. An effective dose may also refer to the amount ofa modified or mutated RSV F protein, an RSV F micelle comprising amodified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein that provides a therapeutic benefit in thetreatment or management of an infection or disease. Further, aneffective dose is the amount with respect to a modified or mutated RSV Fprotein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein of theinvention alone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of an infection ordisease. An effective dose may also be the amount sufficient to enhancea subject's (e.g., a human's) own immune response against a subsequentexposure to an infectious agent or disease. Levels of immunity can bemonitored, e.g., by measuring amounts of neutralizing secretory and/orserum antibodies, e.g., by plaque neutralization, complement fixation,enzyme-linked immunosorbent, or microneutralization assay, or bymeasuring cellular responses, such as, but not limited to cytotoxic Tcells, antigen presenting cells, helper T cells, dendritic cells and/orother cellular responses. T cell responses can be monitored, e.g., bymeasuring, for example, the amount of CD4⁺ and CD8⁺ cells present usingspecific markers by fluorescent flow cytometry or T cell assays, such ashut not limited to T-cell proliferation assay, T-cell cytotoxic assay,TETRAMER assay, and/or ELISPOT assay. In the case of a vaccine, an“effective dose” is one that prevents disease and/or reduces theseverity of symptoms.

As used herein, the term “effective amount” refers to an amount of amodified or mutated RSV F protein, an RSV F micelle comprising amodified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein necessary or sufficient to realize a desiredbiologic effect. An effective amount of the composition would be theamount that achieves a selected result, and such an amount could bedetermined as a matter of routine experimentation by a person skilled inthe art. For example, an effective amount for preventing, treatingand/or ameliorating an infection could be that amount necessary to causeactivation of the immune system, resulting in the development of anantigen specific immune response upon exposure to a modified or mutatedRSV F protein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein of theinvention. The term is also synonymous with “sufficient amount.”

As used herein, the term “expression” refers to the process by whichpolynucleic acids are transcribed into mRNA and translated intopeptides, polypeptides, or proteins. If the polynucleic acid is derivedfrom genomic DNA, expression may, if an appropriate eukaryotic host cellor organism is selected, include splicing of the mRNA. In the context ofthe present invention, the term also encompasses the yield of RSV F genemRNA and RSV F proteins achieved following expression thereof.

As used herein, the term “F protein” or “Fusion protein” or “F proteinpolypeptide” or “Fusion protein polypeptide” refers to a polypeptide orprotein having all or part of an amino acid sequence of an RSV Fusionprotein polypeptide. Similarly, the term “G protein” or “G proteinpolypeptide” refers to a polypeptide or protein having all or part of anamino acid sequence of an RSV Attachment protein polypeptide. NumerousRSV Fusion and Attachment proteins have been described and are known tothose of skill in the art. WO/2008/114149, which is herein incorporatedby reference in its entirety, sets out exemplary F and G proteinvariants (for example, naturally occurring variants

As used herein, the terms “immunogens” or “antigens” refer to substancessuch as proteins, peptides, peptides, nucleic acids that are capable ofeliciting an immune response. Both terms also encompass epitopes, andare used interchangeably.

As used herein the -term “immune stimulator” refers to a compound thatenhances an immune response via the body's own chemical messengers(cytokines). These molecules comprise various cytokines, lymphokines andchemokines with immunostimulatory, immunopotentiating, andpro-inflammatory activities, such as interferons (IFN-γ), interleukins(e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g.,granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and otherimmunostimulatory molecules, such as macrophage inflammatory factor,Flt3 ligand, B7.1; B7.2, etc. The immune stimulator molecules can beadministered in the same formulation as VLPs of the invention, or can beadministered separately. Either the protein or an expression vectorencoding the protein can be administered to produce an immunostimulatoryeffect.

As use herein, the term “immunogenic formulation” refers to apreparation which, when administered to a vertebrate, e.g. a mammal,will induce an immune response.

As use herein, the term “infectious agent” refers to microorganisms thatcause an infection in a vertebrate. Usually, the organisms are viruses,bacteria, parasites, protozoa and/or fungi.

As used herein, the terms “mutated,” “modified,” “mutation,” or“modification” indicate any modification of a nucleic acid and/orpolypeptide which results in an altered nucleic acid or polypeptide.Mutations include, for example, point mutations, deletions, orinsertions of single or multiple residues in a polynucleotide, whichincludes alterations arising within a protein-encoding region of a geneas well as alterations in regions outside of a protein-encodingsequence, such as, but not limited to, regulatory or promoter sequences.A genetic alteration may be a mutation of any type. For instance, themutation may constitute a point mutation, a frame-shift mutation, aninsertion, or a deletion of part or all of a gene. In some embodiments,the mutations are naturally-occurring. In other embodiments, themutations are the results of artificial mutation pressure. In stillother embodiments, the mutations in the RSV F proteins are the result ofgenetic engineering.

As used herein, the term “multivalent” refers to compositions which haveone or more antigenic proteins/peptides or immunogens against multipletypes or strains of infectious agents or diseases.

As used herein, the term “pharmaceutically acceptable vaccine” refers toa formulation which contains a modified or mutated RSV F protein, an RSVF micelle comprising a modified or mutated RSV F protein, or a VLPcomprising a modified or mutated RSV F protein of the present invention,which is in a form that is capable of being administered to a vertebrateand which induces a protective immune response sufficient to induceimmunity to prevent and/or ameliorate an infection or disease, and/or toreduce at least one symptom of an infection or disease, and/or toenhance the efficacy of another dose of a modified or mutated RSV Fprotein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein.Typically, the vaccine comprises a conventional saline or bufferedaqueous solution medium in which the composition of the presentinvention is suspended or dissolved. In this form, the composition ofthe present invention can be used conveniently to prevent, ameliorate,or otherwise treat an infection. Upon introduction into a host, thevaccine is able to provoke an immune response including, but not limitedto, the production of antibodies and/or cytokines and/or the activationof cytotoxic T cells, antigen presenting cells, helper T cells,dendritic cells and/or other cellular responses.

As used herein, the phrase “protective immune response” or “protectiveresponse” refers to an immune response mediated by antibodies against aninfectious agent or disease, which is exhibited by a vertebrate (e.g., ahuman), that prevents or ameliorates an infection or reduces at leastone disease symptom thereof. Modified or mutated RSV F proteins, RSV Fmicelles comprising a modified or mutated RSV F protein, or VLPscomprising a modified or mutated RSV F protein of the invention canstimulate the production of antibodies that, for example, neutralizeinfectious agents, blocks infectious agents from entering cells, blocksreplication of the infectious agents, and/or protect host cells frominfection and destruction. The term can also refer to an immune responsethat is mediated by T-lymphocytes and/or other white blood cells againstan infectious agent or disease; exhibited by a vertebrate a human), thatprevents or ameliorates infection or disease, or reduces at least onesymptom thereof.

As use herein, the term “vertebrate” or “subject” or “patient” refers toany member of the subphylum cordata, including, without limitation,humans and other primates, including non-human primates such aschimpanzees and other apes and monkey species. Farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats (includingcotton rats) and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like are also non-limiting examples . The terms “mammals”and “animals” are included in this definition. Both adult and newbornindividuals are intended to be covered. In particular, infants and youngchildren are appropriate subjects or patients for a RSV vaccine.

As used herein, the term “virus-like particle” (VLP) refers to astructure that in al least one attribute resembles a virus but which hasnot been demonstrated to be infectious. Virus-like particles inaccordance with the invention do not carry genetic information encodingfor the proteins of the virus-like particles. In general, virus-likeparticles lack a viral genome and, therefore, are noninfectious. Inaddition, virus-like particles can often be produced in large quantitiesby heterologous expression and can be easily purified.

As used herein, the term “chimeric VLP” refers to VLPs that containproteins, or portions thereof, from at least two different infectiousagents (heterologous proteins). Usually, one of the proteins is derivedfrom a virus that can drive the formation of VLPs from host cells.Examples, for illustrative purposes, are the BRSV M protein and/or theHRSV G or F proteins. The terms RSV VLPs and chimeric VLPs can be usedinterchangeably where appropriate.

As used herein, the term “vaccine” refers to a preparation of dead orweakened pathogens, or of derived antigenic determinants that is used toinduce formation of antibodies or immunity against the pathogen. Avaccine is given to provide immunity to the disease, for example,influenza, which is caused by influenza viruses. In addition, the term“vaccine” also refers to a suspension or solution of an immunogen (e.g.a modified or mutated RSV F protein, an RSV F micelle comprising amodified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein) that is administered to a vertebrate to produceprotective immunity, i.e., immunity that prevents or reduces theseverity of disease associated with infection. The present inventionprovides for vaccine compositions that are immunogenic and may provideprotection against a disease associated with infection.

RSV F Proteins

Two structural membrane proteins, F and G proteins, are expressed on thesurface of RSV, and have been shown to be targets of neutralizingantibodies (Sullender, W., 2000, Clinical Microbiology Review 13, 1-15).These two proteins are also primarily responsible for viral recognitionand entry into target cells; G protein binds to a specific cellularreceptor and the F protein promotes fusion of the virus with the cell.The F protein is also expressed on the surface of infected cells and isresponsible for subsequent fusion with other cells leading to syncytiaformation. Thus, antibodies to the F protein can neutralize virus orblock entry of the virus into the cell or prevent syncytia formation.Although antigenic and structural differences between A and B subtypeshave been described for both the G and F proteins, the more significantantigenic differences reside on the G protein, where amino acidsequences are only 53% homologous and antigenic relatedness is 5% (Walshet al. (1987) J. Infect. Dis. 155, 1198-1204; and Johnson et at, (1987)Proc. Natl. Acad. Sci. USA 84,5625-5629). Conversely, antibodies raisedto the F protein show a high degree of cross-reactivity among subtype Aand B viruses.

The RSV F protein directs penetration of RSV by fusion between thevirion's envelope protein and the host cell plasma membrane. Later ininfection, the F protein expressed on the cell surface can mediatefusion with neighboring cells to form syncytia. The F protein is a typeI transmembrane surface protein that has a N-terminal cleaved signalpeptide and a membrane anchor near the C-terminus. RSV F is synthesizedas an inactive F₀ precursor that assembles into a homotrimer and isactivated by cleavage in the trans-Golgi complex by a cellularendoprotease to yield two disulfide-linked subunits, F₁ and F₂ subunits,The N-terminus of the F₁ subunit that is created by cleavage contains ahydrophobic domain (the fusion peptide) that inserts directly into thetarget membrane to initiate fusion. The F₁ subunit also contains heptadrepeats that associate during fusion, driving a conformational shiftthat brings the viral and cellular membranes into close proximity(Collins and Crowe, 2007, Fields Virology, 5^(th) ed., D. M Kipe et al.,Lipincott, Williams and Wilkons, p. 1604). SEQ ID NO: 2 (GenBankAccession No. AAB59858) depicts a representative RSV F protein, which isencoded by the gene shown in SEQ ID NO: 1 (GenBank Accession No.M11486).

In nature, the RSV F protein is expressed as a single polypeptideprecursor, 574 amino acids in length, designated FO. In vivo, FOoligomerizes in the endoplasmic reticulum and is proteolyticallyprocessed by a furin protease at two conserved furin consensus sequences(furin cleavage sites), RARR (SEQ ID NO: 23) (secondary) and KKRKRR (SEQID NO: 24) (primary) to generate an oligomer consisting of twodisulfide-linked fragments. The smaller of these fragments is termed F2and originates from the N-terminal portion of the FO precursor. It willbe recognized by those of skill in the art that the abbreviations FO, F1and F2 are commonly designated F₀, F₁ and F₂ in the scientificliterature. The larger, C-terminal F1 fragment anchors the F protein inthe membrane via a sequence of hydrophobic amino acids, which areadjacent to a 24 amino acid cytoplasmic tail. Three F2-F1 dimersassociate to form a mature F protein, which adopts a metastableprefusogenic (“prefusion”) conformation that is triggered to undergo aconformational change upon contact with a target cell membrane. Thisconformational change exposes a hydrophobic sequence, known as thefusion peptide, which associates with the host cell membrane andpromotes fusion of the membrane of the virus, or an infected cell, withthe target cell membrane.

The F1 fragment contains at least two heptad repeat domains, designatedHRA and HRB, and is situated in proximity to the fusion peptide andtransmembrane anchor domains, respectively. In the prefusionconformation, the F2-F1 dimer forms a globular head and stalk structure,in which the HRA domains are in a segmented (extended) conformation inthe globular head. In contrast, the HRB domains form a three-strandedcoiled coil stalk extending from the head region. During transition fromthe prefusion to the postfusion confirmations, the HRA domains collapseand are brought into proximity to the HRB domains to form ananti-parallel six helix bundle. In the postfusion state the fusionpeptide and transmembrane domains are juxtaposed to facilitate membranefusion.

Although the conformational description provided above is based onmolecular modeling of crystallographic data, the structural distinctionsbetween the prefusion and postfusion conformations can be monitoredwithout resort to crystallography. For example, electron micrography canbe used to distinguish between the prefusion and postfusion(alternatively designated prefusogenic and fusogenic) conformations, asdemonstrated by et al., Virology, 271:122-131 (2000) and Morton et al.,Virology, 311: 275-288, which are incorporated herein by reference forthe purpose of their technological teachings. The prefusion conformationcan also be distinguished from the fusogenic (post-fusion) conformationby liposome association assays as described by Connolly et al, Proc.Natl. Acad. Set. USA, 103:17903-17908 (2006), which is also incorporatedherein by reference for the purpose of its technological teachings.Additionally, prefusion and fusogenic conformations can be distinguishedusing antibodies (e.g., monoclonal antibodies) that specificallyrecognize conformation epitopes present on one or the other of theprefusion or fusogenic form of the RSV F protein, but not on the otherform. Such conformation epitopes can be due to preferential exposure ofan antigenic determinant on the surface of the molecule. Alternatively,conformational epitopes can arise from the juxtaposition of amino acidsthat are non-contiguous in the linear polypeptide.

Modified or Mutated RSV F Proteins

The present inventors have found that surprisingly high levels ofexpression of the fusion (F) protein can be achieved when specificmodifications are made to the structure of the RSV F protein. Suchmodifications also unexpectedly reduce the cellular toxicity of the RSVF protein in a host cell. In addition, the modified F proteins of thepresent invention demonstrate an improved ability to exhibit thepost-fusion “lollipop” morphology as opposed to the pre-fusion “rod”morphology. Thus, in one aspect, the modified F proteins of the presentinvention can also exhibit improved (e.g. enhanced) immunogenicity ascompared to wild-type F proteins (e.g. exemplified by SEQ ID NO: 2,which corresponds to GenBank Accession No. AAB59858). Thesemodifications have significant applications to the development ofvaccines and methods of using said vaccines for the treatment and/orprevention of RSV.

In accordance with the invention, any number of mutations can be made tonative or wild-type RSV F proteins, and in a preferred aspect, multiplemutations can be made to result in improved expression and/orimmunogenic properties as compared to native or wild-type RSV Fproteins. Such mutations include point mutations, frame shift mutations,deletions, and insertions, with one or more (e.g., one, two, three, orfour, etc.) mutations preferred.

The native F protein polypeptide can be selected from any F protein ofan RSV A strain, RSV B strain, HRSV A strain, HRSV B strain, BRSVstrain, or avian RSV strain, or from variants thereof (as definedabove). In certain exemplary embodiments, the native F proteinpolypeptide is the F protein represented by SEQ ID NO: 2 (GenBankAccession No AAB59858). To facilitate understanding of this disclosure,all amino acid residue positions, regardless of strain, are given withrespect to (that is, the amino acid residue position corresponds to) theamino acid position of the exemplary F protein. Comparable amino acidpositions of the F protein from other RSV strains can be determinedeasily by those of ordinary skill in the art by aligning the amino acidsequences of the selected RSV strain with that of the exemplary sequenceusing readily available and well-known alignment algorithms (such asBLAST, e.g., using default parameters). Numerous additional examples ofF protein polypeptides from different RSV strains are disclosed inWO/2008/114149 (which is incorporated herein by reference in itsentirety). Additional variants can arise through genetic drift, or canbe produced artificially using site directed or random mutagenesis, orby recombination of two or more preexisting variants. Such additionalvariants are also suitable in the context of the modified or mutated RSVF proteins disclosed herein.

Mutations may be introduced into the RSV F proteins of the presentinvention using any methodology known to those skilled in the art.Mutations may be introduced randomly by, for example, conducting a PCRreaction in the presence of manganese as a divalent metal ion cofactor.Alternatively, oligonucleotide directed mutagenesis may be used tocreate the mutant or modified RSV F proteins which allows for allpossible classes of base pair changes at any determined site along theencoding DNA molecule. In general, this technique involves annealing anoligonucleotide complementary (except for one or more mismatches) to asingle stranded nucleotide sequence coding for the RSV F protein ofinterest. The mismatched oligonucleotide is then extended by DNApolymerase, generating a double-stranded DNA molecule which contains thedesired change in sequence in one strand. The changes in sequence can,for example, result in the deletion, substitution, or insertion of anamino acid. The double-stranded polynucleotide can then be inserted intoan appropriate expression vector, and a mutant or modified polypeptidecan thus be produced. The above-described oligonucleotide directedmutagenesis can, for example, be carried out via PCR.

Additional RSV Proteins

The invention also encompasses RSV virus-like particles (VLPs)comprising a modified or mutated RSV F protein that can be formulatedinto vaccines or antigenic formulations for protecting vertebrates (e.g.humans) against RSV infection or at least one disease symptom thereof.In some embodiments, the VLP comprising a modified or mutated RSV Fprotein further comprises additional RSV proteins, such as M, N, G, andSH. In other embodiments, the VLP comprising a modified or mutated RSV Fprotein further comprises proteins from heterologous strains of virus,such as influenza virus proteins HA, NA, and M1. In one embodiment, theinfluenza virus protein M1 is derived from an avian influenza virusstrain.

RSV N protein binds tightly to both genomic RNA and the replicativeintermediate anti-genomic RNA to form RNAse resistant nucleocapsid. SEQID NOs: 16 (wild-type) and 18 (codon-optimized) depict representativeamino acid sequences of the RSV N protein and SEQ ID NOs: 15 (wild-type)and 17 (codon-optimized) depict representative nucleic acid sequencesencoding the RSV N protein. Encompassed in this invention are RSV Nproteins that are at least about 20%, about 30%, about 40%, about 50%,about 60%, about 70% or about 80%, about 85%, about 90%, about 95%,about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 18,and all fragments and variants (including chimeric proteins) thereof.

RSV M protein is a non-glycosylated internal virion protein thataccumulates in the plasma membrane that interacts with RSV F protein andother factors during virus morphogenesis. In certain preferredembodiments, the RSV M protein is a bovine RSV (BRSV) M protein. SEQ IDNOs: 12 (wild-type) and 14 (codon-optimized) depict representative aminoacid sequences of the BRSV M protein and SEQ ID NOs: 11 (wild-type) and13 (codon-optimized) depict representative nucleic acid sequencesencoding the BRSV M protein. Encompassed in this invention are RSV(including, but not limited to, BRSV) M proteins that are at least about20%, about 30%, about 40%, about 50%, about 60%, about 70% or about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to SEQ ID NOs: 12 and 14, and all fragments andvariants (including chimeric proteins) thereof.

RSV G protein is a type II transmembrane glycoprotein with a singlehydrophobic region near the N-terminal end that serves as both anuncleaved signal peptide and a membrane anchor, leaving the C-terminaltwo-thirds of the molecule oriented externally. RSV G is also expressedas a secreted protein that arises from translational initiation at thesecond AUG in the ORF (at about amino acid 48), which lies within thesignal/anchor. Most of the ectodomain of RSV G is highly divergentbetween RSV strains (Id., p. 1607). SEQ ID NO: 26 depicts arepresentative RSV G protein, which is encoded by the gene sequenceshown in SEQ FD NO: 25. Encompassed in this invention are RSV G proteinsthat are at least about 20%, about 30%, about 40%. about 50%, about 60%,about 70% or about 80%, about 85%, about 90%, about 95%, about 96%,about 97%, about 98% or about 99% identical to SEQ ID NO: 26, and allfragments and variants (including chimeric proteins) thereof.

The SH protein of RSV is a type II transmembrane protein that contains64 (RSV subgroup A) or 65 amino acid residues (RSV subgroup B). Somestudies have suggested that the RSV SH protein may have a role in viralfusion or in changing membrane permeability. However, RSV lacking the SHgene are viable, cause syncytia formation and grow as well as thewild-type virus, indicating that the SH protein is not necessary forvirus entry into host cells or syncytia formation. The SH protein of RSVhas shown the ability of inhibit TNF-α signaling. SEQ ID NO: 27 depictsa representative amino acid sequence of the RSV SH protein. Encompassedin this invention are RSV SH proteins that are at least about 20%, about30%, about 40%, about 50%, about 60%, about 70% or about 80%, about 85%,about 90%, about 95%, about 96%, about 97%, about 98% or about 99%identical to SEQ ID NO: 27, and all fragments and variants (includingchimeric proteins) thereof.

RSV Vaccines

Currently, the only approved approach to prophylaxis of RSV disease ispassive irrrmunization. Initial evidence suggesting a protective rolefor IgG was obtained from observations involving maternal antibody inferrets (Prince, G. A., Ph.D. diss., University of California, LosAngeles, 1975) and humans (Lambrecht et al., (1976) J. Infect. Dis. 134,211-217; and Glezen et al. (1981) J. Pediatr, 98,708-715). Hemming etal. (Morell et al., eds., 1986, Clinical Use of IntravenousImmunoglobulins, Academic Press, London at pages 285-294) recognized thepossible utility of RSV antibody in treatment or prevention of RSVinfection during studies involving the pharmacokinetics of anintravenous immunoglobulin (IVIG) in newborns suspected of havingneonatal sepsis. They noted that one infant, whose respiratorysecretions yielded RSV, recovered rapidly after IVIG infusion.Subsequent analysis of the IVIG lot revealed an unusually high titer ofRSV neutralizing antibody. This same group of investigators thenexamined the ability of hyper-immune serum or immunoglobulin, enrichedfor RSV neutralizing antibody, to protect cotton rats and primatesagainst RSV infection (Prince et al. (1985) Virus Res. 3, 193-206;Prince et al. (1990) J. Virol. 64, 3091-3092. Results of these studiessuggested that RSV neutralizing antibody given prophylacticallyinhibited respiratory tract replication of RSV in cotton rats. Whengiven therapeutically, RSV antibody reduced pulmonary viral replicationboth in cotton rats and in a nonhuman primate model. Furthermore,passive infusion of immune serum or immune globulin did not produceenhanced pulmonary pathology in cotton rats subsequently challenged withRSV.

Since RSV infection can be prevented by providing neutralizingantibodies to a vertebrate, a vaccine comprising a modified or mutatedRSV F protein may induce, when administered to a vertebrate,neutralizing antibodies in vivo. The modified or mutated RSV F proteinsare favorably used for the prevention and/or treatment of RSV infection.Thus, another aspect of this disclosure concerns a method for elicitingan immune response against RSV. The method involves administering animmunologically effective amount of a composition containing a modifiedor mutated RSV F protein to a subject (such as a human or animalsubject). Administration of an immunologically effective amount of thecomposition elicits an immune response specific for epitopes present onthe modified or mutated RSV F protein. Such an immune response caninclude B cell responses (e.g., the production of neutralizingantibodies) and/or T cell responses (e.g., the production of cytokines).Preferably, the immune response elicited by the modified or mutated RSVF protein includes elements that are specific for at least oneconformational epitope present on the modified or mutated RSV F protein.In one embodiment, the immune response is specific for an epitopepresent on an RSV F protein found in the “lollipop” post-fusion activestate. The RSV F proteins and compositions can be administered to asubject without enhancing viral disease following contact with RSV.Preferably, the modified or mutated RSV F proteins disclosed herein andsuitably formulated immunogenic compositions elicit a Th1 biased immuneresponse that reduces or prevents infection with a RSV and/or reduces orprevents a pathological response following infection with a RSV.

In one embodiment, the RSV F proteins of the present invention are foundin the form of micelles (e.g. rosettes). The micelles obtainable inaccordance with the invention consist of aggregates of theimmunogenically active F spike proteins having a rosette-like structure.The rosettes are visible in the electron microscope (Calder et cal.,2000. Virology 271: 122-131). Preferably, the micelles of the presentinvention comprising modified or mutated RSV F proteins exhibit the“lollipop” morphology indicative of the post-fusion active state. In oneembodiment, the micelles are purified following expression in a hostcell. When administered to a subject, the micelles of the presentinvention preferably induce neutralizing antibodies. In someembodiments, the micelles may be administered with an adjuvant. In otherembodiments, the micelles may be administered without an adjuvant.

In another embodiment, the invention encompasses RSV virus-likeparticles (VLPs) comprising a modified or mutated RSV F protein that canbe formulated into vaccines or antigenic formulations for protectingvertebrates (e.g. humans) against RSV infection or at least one diseasesymptom thereof. The present invention also relates to RSV VLPs andvectors comprising wild-type and mutated RSV genes or a combinationthereof derived from different strains of RSV virus, which whentransfected into host cells, will produce virus like particles (VLPs)comprising RSV proteins.

In some embodiments, RSV virus-like particles may further comprise atleast one viral matrix protein (e.g. an RSV M protein). In oneembodiment, the M protein is derived from a human strain of RSV. Inanother embodiment, the M protein is derived from a bovine strain ofRSV. In other embodiments, the matrix protein may be an M1 protein froma strain of influenza virus. In one embodiment, the strain of influenzavirus is an avian influenza strain. In a preferred embodiment, the avianinfluenza strain is the H5N1 strain A/Indonesia/5/05. In otherembodiments, the matrix protein may be from Newcastle Disease Virus(NDV).

In some embodiments, the VLPs may further comprise an RSV G protein. Inone embodiment, the G protein may be from HRSV group A. In anotherembodiment, the G protein may be from HRSV group B. In yet anotherembodiment, the RSV G may be derived from HRSV group A and/or group B.

In some embodiments, the VLPs may further comprise an RSV SH protein. Inone embodiment, the SH protein may be from HRSV group A. In anotherembodiment, the SH protein may be from HRSV group B. In yet anotherembodiment, the RSV SH may be derived from HRSV group A and/or group B.

In some embodiments, VLPs may further comprise an RSV N protein. In oneembodiment, the N protein may be from HRSV group A. In anotherembodiment, the N protein may be from HRSV group B. In yet anotherembodiment, the RSV N may be derived from HRSV group A and/or group B.

In further embodiments, VLPs of the invention may comprise one or moreheterologous immunogens, such as influenza hemagglutinin (HA) and/orneuraminidase (NA).

In some embodiments, the invention also comprises combinations ofdifferent RSV M, F, N, SH, and/or G proteins from the same and/ordifferent strains in one or more VLPs. In addition, the VLPs can includeone or more additional molecules for the enhancement of an immuneresponse.

In another embodiment of the invention, the RSV VLPs can carry agentssuch as nucleic acids, siRNA, micro-RNA, chemotherapeutic agents,imaging agents, and/or other agents that need to be delivered to apatient.

VLPs of the invention are useful for preparing vaccines and immunogeniccompositions. One important feature of VLPs is the ability to expresssurface proteins of interest so that the immune system of a vertebrateinduces an immune response against the protein of interest. However, notall proteins can be expressed on the surface of VLPs. There may be manyreasons why certain proteins are not expressed, or be poorly expressed,on the surface of VLPs. One reason is that the protein is not directedto the membrane of a host cell or that the protein does not have atransmembrane domain. As an example, sequences near the carboxylterminus of influenza hemagglutinin may be important for incorporationof HA into the lipid Mayer of the mature influenza envelopednucleocapsids and for the assembly of HA trimer interaction with theinfluenza matrix protein M1 (Ali, et at., (2000) J. Virol. 74, 8709-19).

Thus, one embodiment of the invention comprises chimeric VLPs comprisinga modified or mutated F protein from RSV and at least one immunogenwhich is not normally efficiently expressed on the cell surface or isnot a normal RSV protein. In one embodiment, the modified or mutated RSVF protein may be fused with an immunogen of interest. In anotherembodiment, the modified or mutated RSV F protein associates with theimmunogen via the transmembrane domain and cytoplasmic tail of aheterologous viral surface membrane protein, e.g., MMTV envelopeprotein.

Other chimeric VLPs of the invention comprise VLPs comprising a modifiedor mutated RSV F protein and at least one protein from a heterologousinfectious agent. Examples of heterologous infectious agent include butare not limited to a virus, a bacterium, a protozoan, a fungi and/or aparasite. In one embodiment, the immunogen from another infectious agentis a heterologous viral protein. In another embodiment, the protein froma heterologous infectious agent is an envelope-associated protein. Inanother embodiment, the protein from another heterologous infectiousagent is expressed on the surface of VLPs. In another embodiment, theprotein from an infectious agent comprises an epitope that will generatea protective immune response in a vertebrate. In one embodiment, theprotein from another infectious agent is co-expressed with a modified ormutated RSV F protein. In another embodiment, the protein from anotherinfectious agent is fused to a modified or mutated RSV F protein. Inanother embodiment, only a portion of a protein from another infectiousagent is fused to a modified or mutated RSV F protein. In anotherembodiment, only a portion of a protein from another infectious agent isfused to a portion of a modified or mutated RSV F protein. In anotherembodiment, the portion of the protein from another infectious agentfused to modified or mutated RSV F protein is expressed on the surfaceof VLPs.

The invention also encompasses variants of the proteins expressed on orin the VLPs of the invention. The variants may contain alterations inthe amino acid sequences of the constituent proteins. The term “variant”with respect to a protein refers to an amino acid sequence that isaltered by one or more amino acids with respect to a reference sequence.The variant can have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties, e.g., replacement ofleucine with isoleucine. Alternatively, a variant can have“nonconservative” changes, e.g., replacement of a glycine with atryptophan. Analogous minor variations can also include amino aciddeletion or insertion, or both. Guidance in determining which amino acidresidues can be substituted, inserted, or deleted without eliminatingbiological or immunological activity can be found using computerprograms well known in the art, for example, DNASTAR software.

Natural variants can occur due to mutations in the proteins. Thesemutations may lead to antigenic variability within individual groups ofinfectious agents, for example influenza. Thus, a person infected with,for example, an influenza strain develops antibody against that virus,as newer virus strains appear, the antibodies against the older strainsno longer recognize the newer virus and re-infection can occur. Theinvention encompasses all antigenic and genetic variability of proteinsfrom infectious agents for making VLPs.

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, cellculture and the like, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger) Sambrook et al., Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (“Ausubel”). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, e.g., thecloning and mutating and/or G molecules of RSV, etc. Thus, the inventionalso encompasses using known methods of protein engineering andrecombinant DNA technology to improve or alter the characteristics ofthe proteins expressed on or in the VLPs of the invention. Various typesof mutagenesis can be used to produce and/or isolate variant nucleicacids that encode for protein molecules and/or to further modify/mutatethe proteins in or on the VLPs of the invention. They include but arenot limited to site-directed, random point mutagenesis, homologousrecombination (DNA shuffling), mutagenesis using uracil containingtemplates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

The invention further comprises protein variants which show substantialbiological activity, e.g., able to elicit an effective antibody responsewhen expressed on or in VLPs of the invention. Such variants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as have little effect onactivity.

Methods of cloning the proteins are known in the art. For example, thegene encoding a specific RSV protein can be isolated by RT-PCR frompolyadenylated mRNA extracted from cells which had been infected with aRSV virus. The resulting product gene can be cloned as a DNA insert intoa vector. The term “vector” refers to the means by which a nucleic acidcan be propagated and/or transferred between organisms, cells, orcellular components. Vectors include plasmids, viruses, bacteriophages,pro-viruses, phagemids, transposons, artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that is not autonomously replicating. In many, but not all, commonembodiments, the vectors of the present invention are plasmids orbacmids.

Thus, the invention comprises nucleotides that encode proteins,including chimeric molecules, cloned into an expression vector that canbe expressed in a cell that induces the formation of VLPs of theinvention. An “expression vector” is a vector, such as a plasmid that iscapable of promoting expression, as well as replication of a nucleicacid incorporated therein. Typically, the nucleic acid to be expressedis “operably linked” to a promoter and/or enhancer, and is subject totranscription regulatory control by the promoter and/or enhancer. In oneembodiment, the nucleotides encode for a modified or mutated RSV Fprotein (as discussed above). In another embodiment, the vector furthercomprises nucleotides that encode the M and/or G RSV proteins. Inanother embodiment, the vector further comprises nucleotides that encodethe M and/or N RSV proteins. In another embodiment, the vector furthercomprises nucleotides that encode the M, G and/or N RSV proteins. Inanother embodiment, the vector further comprises nucleotides that encodea BRSV M protein and/or N RSV proteins. In another embodiment, thevector further comprises nucleotides that encode a BRSV M and/or Gprotein, or influenza HA and/or NA protein. In another embodiment, thenucleotides encode a modified or mutated RSV F and/or RSV G protein withan influenza HA and/or NA protein. In another embodiment, the expressionvector is a baculovirus vector.

In some embodiments of the invention, proteins may comprise mutationscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedprotein or how the proteins are made. Nucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the human mRNA to thosepreferred by insect cells such as Sf9 cells. See U.S. Patent Publication2005/0118191, herein incorporated by reference in its entirely for allpurposes.

In addition, the nucleotides can be sequenced to ensure that the correctcoding regions were cloned and do not contain any unwanted mutations.The nucleotides can be subcloned into an expression vector (e.g.baculovirus) for expression in any cell. The above is only one exampleof how the RSV viral proteins can be cloned. A person with skill in theart understands that additional methods are available and are possible.

The invention also provides for constructs and/or vectors that compriseRSV nucleotides that encode for RSV structural genes, including F, M, G,N, SH, or portions thereof, and/or any chimeric molecule describedabove. The vector may be, for example, a phage, plasmid, viral, orretro-viral vector. The constructs and/or vectors that comprise RSVstructural genes, including F, M, G, N, SH, or portions thereof, and/orany chimeric molecule described above, should be operatively linked toan appropriate promoter, such as the AcMNPV polyhedrin promoter (orother baculovirus), phage lambda PL promoter, the E. coli lac, phoA andtac promoters, the SV40 early and late promoters, and promoters ofretroviral LTRs are non-limiting examples. Other suitable promoters willbe known to the skilled artisan depending on the host cell and/or therate of expression desired. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome-binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon appropriately positioned at the end of the polypeptideto be translated.

Expression vectors will preferably include at least one selectablemarker. Such markers include dihydrofolate reductase, G418 or neomycinresistance for eukaryotic cell culture and tetracycline, kanamycin orampicillin resistance genes for culturing in E. coli and other bacteria.Among vectors preferred are virus vectors, such as baculovirus, poxvirus(e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus,raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canineadenovirus), herpesvirus, and retrovirus. Other vectors that can be usedwith the invention comprise vectors for use in bacteria, which comprisepQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A,pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRlT5.Among preferred eukaryotic vectors are pFastBac1 pWINEO, pSV2CAT, pOG44,pXT1 and pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors willbe readily apparent to the skilled artisan. In one embodiment, thevector that comprises nucleotides encoding for RSV genes, includingmodified or mutated RSV F genes, as well as genes for M, G, N, SH orportions thereof, and/or any chimeric molecule described above, ispFastBac.

The recombinant constructs mentioned above could be used to transfect,infect, or transform and can express RSV proteins, including a modifiedor mutated RSV F protein and at least one immunogen. In one embodiment,the recombinant construct comprises a modified or mutated RSV F, M, G,N, SH, or portions thereof, and/or any molecule described above, intoeukaryotic cells and/or prokaryotic cells. Thus, the invention providesfor host cells which comprise a vector (or vectors) that contain nucleicacids which code for RSV structural genes, including a modified ormutated RSV F; and at least one immunogen such as but not limited to RSVG, N, and SH, or portions thereof, and/or any molecule described above,and permit the expression of genes, including RSV F, G, N, M, or SH orportions thereof, and/or any molecule described above in the host cellunder conditions which allow the formation of VLPs.

Among eukaryotic host cells are yeast, insect, avian, plant, C. elegans(or nematode) and mammalian host cells. Non limiting examples of insectcells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21,Trichoplusia ni cells, e.g. High Five cells, and Drosophila S2 cells.Examples of fungi (including yeast) host cells are S. cerevisiae,Kluyveromyces lactis (K. lacus), species of Candida including C.albicans and C. glabraia, Aspergillus nidulans, Schizosaccharomycespombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples ofmammalian cells are COS cells, baby hamster kidney cells, mouse L cells,LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney(HEK) cells, and African green monkey cells, CV1 cells, HeLa cells, MDCKcells, Vero and Hep-2 cells. Xenopus laevis oocytes, or other cells ofamphibian origin, may also be used. Examples of prokaryotic host cellsinclude bacterial cells, for example, E. coli, B. subtilis, Salmonellatyphi and mycobacteria.

Vectors, e.g., vectors comprising polynucleotides of a modified ormutated RSV F protein; and at least one immunogen including but notlimited to RSV G, N, or SH or portions thereof, and/or any chimericmolecule described above, can be transfected into host cells accordingto methods well known in the art. For example, introducing nucleic acidsinto eukaryotic cells can be by calcium phosphate co-precipitation,electroporation, microinjection, lipofection, and transfection employingpolyamine transfection reagents. In one embodiment, the vector is arecombinant baculovirus. In another embodiment, the recombinantbaculovirus is transfected into a eukaryotic cell. In a preferredembodiment, the cell is an insect cell. In another embodiment, theinsect cell is a Sf9 cell.

This invention also provides for constructs and methods that willincrease the efficiency of VLP production. For example, the addition ofleader sequences to the RSV F, M, G, N, SH, or portions thereof, and/orany chimeric air heterologous molecules described above, can improve theefficiency of protein transporting within the cell. For example, aheterologous signal sequence can be fused to the F, M, G, N, SH, orportions thereof, and/or any chimeric or heterologous molecule describedabove. In one embodiment, the signal sequence can be derived from thegene of an insect cell and fused to M, F, G, N, SH, or portions thereof,and/or any chimeric or heterologous molecules described above. Inanother embodiment, the signal peptide is the chitinase signal sequence,which works efficiently in baculovirus expression systems.

Another method to increase efficiency of VLP production is to codonoptimize the nucleotides that encode RSV including a modified or mutatedRSV F protein, M, G, N, SH or portions thereof, and/or any chimeric orheterologous molecules described above for a specific cell type. Forexamples of codon optimizing nucleic acids for expression in Sf9 cellsee SEQ ID Nos: 3, 5, 7, 9, 13, 17, 19, and 25.

The invention also provides for methods of producing VLPs, the methodscomprising expressing RSV genes including a modified or mutated RSV Fprotein, and at least one additional protein, including but not limitedto RSV M, G, N, SH, or portions thereof, and/or any chimeric orheterologous molecules described above under conditions that allow VLPformation. Depending on the expression system and host cell selected,the VLPs are produced by growing host cells transformed by an expressionvector under conditions whereby the recombinant proteins are expressedand VLPs are formed. In one embodiment, the invention comprises a methodof producing a VLP, comprising transfecting vectors encoding at leastone modified or mutated RSV F protein into a suitable host cell andexpressing the modified or mutated RSV F protein under conditions thatallow VLP formation. In another embodiment, the eukaryotic cell isselected from the group consisting of, yeast, insect, amphibian, avianor mammalian cells. The selection of the appropriate growth conditionsis within the skill or a person with skill of one of ordinary skill inthe art.

Methods to grow cells engineered to produce VLPs of the inventioninclude, but are not limited to, batch, batch-fed, continuous andperfusion cell culture techniques. Cell culture means the growth andpropagation of cells in a bioreactor (a fermentation chamber) wherecells propagate and express protein (e.g. recombinant proteins) forpurification and isolation. Typically, cell culture is performed understerile, controlled temperature and atmospheric conditions in abioreactor. A bioreactor is a chamber used to culture cells in whichenvironmental conditions such as temperature, atmosphere, agitationand/or pH can be monitored. In one embodiment, the bioreactor is astainless steel chamber. In another embodiment, the bioreactor is apre-sterilized plastic bag (e.g. Cellbag®, Wave Biotech, Bridgewater,N.J.). In other embodiment, the pre-sterilized plastic bags are about 50L to 1000 L bags.

The VLPs are then isolated using methods that preserve the integritythereof such as by gradient centrifugation, e.g., cesium chloride,sucrose and iodixanol, as well as standard purification techniquesincluding, e.g., ion exchange and gel filtration chromatography.

The following is an example of how VLPs of the invention can be made,isolated and purified. Usually VLPs are produced from recombinant celllines engineered to create VLPs when the cells are grown in cell culture(see above). A person of skill in the art would understand that thereare additional methods that can be utilized to make and purify VLPs ofthe invention, thus the invention is not limited to the methoddescribed.

Production of VLPs of the invention can start by seeding Sf9 cells(non-infected) into shaker flasks, allowing the cells to expand andscaling up as the cells grow and multiply (for example from a 125-mlflask to a 50 L Wave bag). The medium used to grow the cell isformulated for the appropriate cell line (preferably serum free media,e.g. insect medium ExCell-420, JRH). Next, the cells are infected withrecombinant baculovirus at the most efficient multiplicity of infection(e.g. from about 1 to about 3 plaque forming units per cell). Onceinfection has occurred, the modified or mutated RSV F protein, M, G, N,SH, or portions thereof, and/or any chimeric or heterologous moleculedescribed above, are expressed from the virus genome, self assemble intoVLPs and are secreted from the cells approximately 24 to 72 hours postinfection. Usually, infection is most efficient when the cells are inmid-log phase of growth (4-8×10⁶ cells/ml) and are at least about 90%viable.

VLPs of the invention can be harvested approximately 48 to 96 hours postinfection, when the levels of VLPs in the cell culture medium are nearthe maximum but before extensive cell lysis. The Sf9 cell density andviability at the time of harvest can be about 0.5'10⁶ cells/ml to about1.5×10⁶ cells/ml with at least 20% viability, as shown by dye exclusionassay. Next, the medium is removed and clarified. NaCl can be added tothe medium to a concentration of about 0.4 to about 1.0 M, preferably toabout 0.5 M, to avoid VLP aggregation. The removal of cell and cellulardebris from the cell culture medium containing VLPs of the invention canbe accomplished by tangential flow filtration (TFF) with a single use,pre-sterilized hollow fiber 0.5 or 1.00 μm filter cartridge or a similardevice.

Next, VLPs in the clarified culture medium can be concentrated byultra-filtration using a disposable, pre-sterilized 500,000 molecularweight cut off hollow fiber cartridge. The concentrated VLPs can bediafiltrated against 10 volumes pH 7.0 to 8.0 phosphate-buffered saline(PBS) containing 0.5 M NaCl to remove residual medium components.

The concentrated, diafiltered VLPs can be furthered purified on a 20% to60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaClby centrifugation at 6,500×g for 18 hours at about 4° C. to about 10° C.Usually VLPs will form a distinctive visible band between about 30% toabout 40% sucrose or at the interface (in a 20% and 60% step gradient)that can be collected from the gradient and stored. This product can bediluted to comprise 200 mM of NaCl in preparation for the next step inthe purification process. This product contains VLPs and may containintact baculovirus particles.

Further purification of VLPs can be achieved by anion exchangechromatography, or 44% isopycnic sucrose cushion centrifugation. Inanion exchange chromatography, the sample from the sucrose gradient (seeabove) is loaded into column containing a medium with an anion (e.g.Matrix Fractolzel EMD TMAE) and eluded via a salt gradient (from about0.2 M to about 1.0 M of NaCl) that can separate the VLP from othercontaminates (e.g. baculovirus and DNA/RNA). In the sucrose cushionmethod, the sample comprising the VLPs is added to a 44% sucrose cushionand centrifuged for about 18 hours at 30,000 g. VLPs form a band at thetop of 44% sucrose, while baculovirus precipitates at the bottom andother contaminating proteins stay in the 0% sucrose layer at the top.The VLP peak or band is collected.

The intact baculovirus can be inactivated, if desired. Inactivation canbe accomplished by chemical methods, for example, formalin orβ-propiolactone (BPL). Removal and/or inactivation of intact baculoviruscan also be largely accomplished by using selective precipitation andchromatographic methods known in the art, as exemplified above. Methodsof inactivation comprise incubating the sample containing the VLPs in0.2% of BPL for 3 hours at about 25° C. to about 27° C. The baculoviruscan also be inactivated by incubating the sample containing the VLPs at0.05% BPL at 4° C. for 3 days, then at 37° C. for one hour.

After the inactivation/removal step, the product comprising VLPs can berun through another diafiltration step to remove any reagent from theinactivation step and/or any residual sucrose, and to place the VLPsinto the desired buffer (e.g. PBS). The solution comprising VLPs can besterilized by methods known in the art (e.g. sterile filtration) andstored in the refrnzerator or freezer.

The above techniques can be practiced across a variety of scales. Forexample, T-flasks, shake-flasks, spinner battles, up to industrial sizedbioreactors. The bioreactors can comprise either a stainless steel tankor a pre-sterilized plastic bag (for example, the system sold by WaveBiotech, Bridgewater, N.J.). A person with skill in the art will knowwhat is most desirable for their purposes.

Expansion and production of baculovirus expression vectors and infectionof cells with recombinant baculovirus to produce recombinant RSV VLPscan be accomplished in insect cells, for example SP9 insect cells aspreviously described. In one embodiment, the cells are SF9 infected withrecombinant baculovirus engineered to produce RSV VLPs.

Pharmaceutical or Vaccine Formulations and Administration

The pharmaceutical compositions useful herein contain a pharmaceuticallyacceptable carrier, including any suitable diluent or excipient, whichincludes any pharmaceutical agent that does not itself induce theproduction of an immune response harmful to the vertebrate receiving thecomposition, and which may be administered without undue toxicity and a.modified or mutated RSV F protein, an RSV F micelle comprising amodified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein of the invention. As used herein, the term“pharmaceutically acceptable” means being approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopia, European Pharmacopia or other generally recognizedpharmacopia for use in mammals, and more particularly in humans. Thesecompositions can be useful as a vaccine and/or antigenic compositionsfor inducing a protective immune response in a vertebrate.

The invention encompasses a pharmaceutically acceptable vaccinecomposition comprising VLPs comprising at least one modified or mutatedRSV F protein, and at least one additional protein, including but notlimited to RSV M, G, N, SH, or portions thereof, and/or any chimeric orheterologous molecules described above. In one embodiment, thepharmaceutically acceptable vaccine composition comprises VLPscomprising at least one modified or mutated RSV F protein and at leastone additional immunogen. In another embodiment, the pharmaceuticallyacceptable vaccine composition comprises VLPs comprising at least onemodified or mutated RSV F protein and at least one RSV M protein. Inanother embodiment, the pharmaceutically acceptable vaccine compositioncomprises VLPs comprising at least one modified or mutated. RSV Fprotein and at least one BRSV M protein. In another embodiment, thepharmaceutically acceptable vaccine composition comprises VLPscomprising at least one modified or mutated RSV F protein and at leastone influenza M1 protein. In another embodiment, the pharmaceuticallyacceptable vaccine composition comprises VLPs comprising at least onemodified or mutated RSV F protein and at least one avian influenza M1protein.

In another embodiment, the pharmaceutically acceptable vaccinecomposition comprises VLPs further comprising an RSV G protein,including but not limited to a HRSV, BRSV or avian RSV G protein. Inanother embodiment, the pharmaceutically acceptable vaccine compositioncomprises VLPs further comprising RSV N protein, including but notlimited to a HRSV, BRSV or avian RSV N protein, In another embodiment,the pharmaceutically acceptable vaccine composition comprises VLPsfurther comprising RSV SH protein, including but not limited to a HRSV,BRSV or avian RSV SH protein.

In another embodiment, the invention encompasses a pharmaceuticallyacceptable vaccine composition comprising chimeric VLPs such as VLPscomprising BRSV M and a modified or mutated RSV F protein and/or G, H,or SH protein from a RSV and optionally HA or NA protein derived from aninfluenza virus, wherein the HA or NA protein is a fused to thetransmembrane domain and cytoplasmic tail of RSV F and/or G protein.101431 The invention also encompasses a pharmaceutically acceptablevaccine composition comprising modified or mutated RSV F protein, an RSVF micelle comprising a modified or mutated RSV F protein, or a VLPcomprising a modified or mutated RSV F protein as described above.

In one embodiment, the pharmaceutically acceptable vaccine compositioncomprises VIPs comprising a modified or mutated RSV F protein and atleast one additional protein. In another embodiment, thepharmaceutically acceptable vaccine composition comprises VLPs furthercomprising RSV M protein, such as but not limited to a BRSV M protein.In another embodiment, the pharmaceutically acceptable vaccinecomposition comprises VLPs further comprising RSV G protein, includingbut not limited to a HRSV G protein. In another embodiment, thepharmaceutically acceptable vaccine composition comprises VLPs furthercomprising RSV N protein, including but not limited to a HRSV, BRSV oravian RSV N protein. In another embodiment, the pharmaceuticallyacceptable vaccine composition comprises VLPs further comprising RSV SHprotein, including but not limited to a HRSV, BRSV or avian RSV SHprotein. In another embodiment, the pharmaceutically acceptable vaccinecomposition comprises VLPs comprising BRSV M protein and F and/or Gprotein from HRSV group A. In another embodiment, the pharmaceuticallyacceptable vaccine composition comprises VLPs comprising BRSV M proteinand F and/or G protein from HRSV group B. In another embodiment, theinvention encompasses a pharmaceutically acceptable vaccine compositioncomprising chimeric VLPs such as VLPs comprising chimeric M protein froma BRSV and optionally HA protein derived from an influenza virus,wherein the M protein is fused to the influenza. HA protein. In anotherembodiment, the invention encompasses a pharmaceutically acceptablevaccine composition comprising chimeric VLPs such as VLPs comprisingBRSV M, and a chimeric F and/or G protein from a RSV and optionally HAprotein derived from an influenza virus, wherein the chimeric influenzaHA protein is fused to the transmembrane domain and cytoplasmic tail ofRSV F and/or G protein. In another embodiment, the invention encompassesa pharmaceutically acceptable vaccine composition comprising chimericVLPs such as VLPs comprising BRSV M and a chimeric F and/or G proteinfrom a RSV and optionally HA or NA protein derived from an influenzavirus, wherein the HA or NA protein is a fused to the transmetnbranedomain and cytoplasmic tail of RSV F and/or G protein.

The invention also encompasses a pharmaceutically acceptable vaccinecomposition comprising a chimeric VLP that comprises at least one RSVprotein. In one embodiment, the pharmaceutically acceptable vaccinecomposition comprises VLPs comprising a modified or mutated RSV Fprotein and at least one immunogen from a heterologous infectious agentor diseased cell. In another embodiment, the immunogen from aheterologous infectious agent is a viral protein. In another embodiment,the viral protein from a heterologous infectious agent is an envelopeassociated protein. In another embodiment, the viral protein from aheterologous infectious agent is expressed on the surface of VLPs. Inanother embodiment, the protein from an infectious agent comprises anepitope that will generate a protective immune response in a vertebrate.

The invention also encompasses a kit for immunizing a vertebrate, suchas a human subject, comprising VLPs that comprise at least one RSVprotein. In one embodiment, the kit comprises VLPs comprising a modifiedor mutated RSV F protein. In one embodiment, the kit further comprises aRSV M protein such as a BRSV M protein. In another embodiment, the kitfurther comprises a RSV G protein. In another embodiment, the inventionencompasses a kit comprising VLPs which comprises a chimeric M proteinfrom a BRSV and optionally HA protein derived from an influenza virus,wherein the M protein is fused to the BRSV M. In another embodiment, theinvention encompasses a kit comprising VLPs which comprises a chimeric Mprotein from a BRSV, a RSV F and/or G protein and an immunogen from aheterologous infectious agent. In another embodiment, the inventionencompasses a kit comprising VLPs which comprises a M protein from aBRSV, a chimeric RSV F and/or G protein and optionally HA proteinderived from an influenza virus, wherein the HA protein is fused to thetransmembrane domain and cytoplasmic tail of RSV F or G protein. Inanother embodiment, the invention encompasses a kit comprising VLPswhich comprises M protein from a BRSV, a chimeric RSV F and/or G proteinand optionally HA or NA protein derived from an influenza virus, whereinthe HA protein is fused to the transmembrane domain and cytoplasmic tailof RSV F and/or G protein.

In one embodiment, the invention comprises an immunogenic formulationcomprising at least one effective dose of a modified or mutated RSV Fprotein. In another embodiment, the invention comprises an immunogenicformulation comprising at least one effective dose of an RSV F micellecomprising a modified or mutated RSV F protein, In yet anotherembodiment, the invention comprises an immunogenic formulationcomprising at least one effective dose of a VLP comprising a modified ormutated RSV F protein as described above.

The immunogenic formulation of the invention comprises a modified ormutated RSV F protein, an RSV F micelle comprising a modified or mutatedRSV F protein, or a VLP comprising a modified or mutated RSV F protein,and a pharmaceutically acceptable carrier or excipient. Pharmaceuticallyacceptable carriers include but are not limited to saline, bufferedsaline, dextrose, water, glycerol, sterile isotonic aqueous buffer, andcombinations thereof. A thorough discussion of pharmaceuticallyacceptable carriers, diluents, and other excipients is presented inRemington's Pharmaceutical Sciences (Mack Pub. Co. N.J. currentedition). The formulation should suit the mode of administration, in apreferred embodiment, the formulation is suitable for administration tohumans, preferably is sterile, non-particulate and/or non-pyrogenic.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be asolid form, such as a lyophilized powder suitable for reconstitution, aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc.

The invention also provides for a pharmaceutical pack or kit comprisingone or more containers filled with one or more of the ingredients of thevaccine formulations of the invention. In a preferred embodiment, thekit comprises two containers, one containing a modified or mutated RSV Fprotein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein, andthe other containing an adjuvant. Associated with such container(s) canbe a notice in the form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

The invention also provides that the formulation be packaged in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of composition. In one embodiment, the composition issupplied as a liquid, in another embodiment, as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted, e.g., with water or saline to theappropriate concentration for administration to a subject.

In an alternative embodiment, the composition is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the composition. Preferably, the liquid form of thecomposition is supplied in a hermetically sealed container at leastabout 50 μg/ml, more preferably at least about 100 μg/ml, at least about200 μg/ml, at least 500 μg/ml, or at least 1 mg/ml.

As an example, chimeric RSV VLPs comprising a modified or mutated RSV Fprotein of the invention are administered in an effective amount orquantity (as defined above) sufficient to stimulate an immune response,each a response against one or more strains of RSV. Administration ofthe modified or mutated RSV F protein, an RSV F micelle comprising amodified or mutated RSV F protein, or VLP of the invention elicitsimmunity against RSV. Typically, the dose can be adjusted within thisrange based on, e.g., age, physical condition, body weight, sex, diet,time of administration, and other clinical factors. The prophylacticvaccine formulation is systemically administered, e.g., by subcutaneousor intramuscular injection using a needle and syringe, or a needle-lessinjection device. Alternatively, the vaccine formulation is administeredintranasally, either by drops, large particle aerosol (greater thanabout 10 microns), or spray into the upper respiratory tract. While anyof the above routes of delivery results in an immune response,intranasal administration confers the added benefit of eliciting mucosalimmunity at the site of entry of many viruses, including RSV andinfluenza.

Thus, the invention also comprises a method of formulating a vaccine orantigenic composition that induces immunity to an infection or at leastone disease symptom thereof to a mammal, comprising adding to theformulation an effective dose of a modified or mutated RSV F protein, anRSV F micelle comprising a modified or mutated RSV F protein, or a VLPcomprising a modified or mutated RSV F protein. In one embodiment, theinfection is an RSV infection.

While stimulation of immunity with a single dose is possible, additionaldosages can be administered, by the same or different route, to achievethe desired effect. In neonates and infants, for example, multipleadministrations may be required to elicit sufficient levels of immunity.Administration can continue at intervals throughout childhood, asnecessary to maintain sufficient levels of protection againstinfections, e.g. RSV infection. Similarly, adults who are particularlysusceptible to repeated or serious infections, such as, for example,health care workers, day care workers, family members of young children,the elderly, and individuals with compromised cardiopulmonary functionmay require multiple immunizations to establish and/or maintainprotective immune response Levels of induced immunity can be monitored,for example, by measuring amounts of neutralizing secretory and serumantibodies, and dosages adjusted or vaccinations repeated as necessaryto elicit and maintain desired levels of protection.

Methods of administering a composition comprising a modified or mutatedRSV F protein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein (e.g.vaccine and/or antigenic formulations) include, but are not limited to,parenteral administration (e.g., intradermal, intramuscular, intravenousand subcutaneous), epidural, and mucosal (e.g., intranasal and oral orpulmonary routes or by suppositories). In a specific embodiment,compositions of the present invention are administered intramuscularly,intravenously, subcutaneously, transdermally or intradermally. Thecompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucous, colon, conjunctiva,nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. In some embodiments, intranasal or other mucosal routesof administration of a composition of the invention may induce anantibody or other immune response that is substantially higher thanother routes of administration. In another embodiment, intranasal orother mucosal routes of administration of a composition of the inventionmay induce an antibody or other immune response that will induce crossprotection against other strains of RSV. Administration can be systemicor local.

In yet another embodiment, the vaccine and/or immunogenic formulation isadministered in such a manner as to target mucosal tissues in order toelicit an immune response at the site of immunization. For example,mucosal tissues such as gut associated lymphoid tissue (GALT) can betargeted for immunization by using oral administration of compositionswhich contain adjuvants with particular mucosal targeting properties.Additional mucosal tissues can also be targeted, such as nasopharyngeallymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT).

Vaccines and/or immunogenic formulations of the invention may also beadministered on a dosage schedule, for example, an initialadministration of the vaccine composition with subsequent boosteradministrations. In particular embodiments, a second dose of thecomposition is administered anywhere from two weeks to one year,preferably from about 1, about 2, about 3, about 4, about 5 to about 6months, after the initial administration. Additionally, a third dose maybe administered after the second dose and from about three months toabout two years, or even longer, preferably about 4, about 5, or about 6months, or about 7 months to about one year after the initialadministration. The third dose may be optionally administered when no orlow levels of specific immunoglobulins are detected in the serum and/orurine or mucosal secretions of the subject after the second dose. In apreferred embodiment, a second dose is administered about one monthafter the first administration and a third dose is administered aboutsix months after the first administration. In another embodiment, thesecond dose is administered about six months after the firstadministration. In another embodiment, the compositions of the inventioncan be administered as part of a combination therapy. For example,compositions of the invention can be formulated with other immunogeniccompositions, antivirals and/or antibiotics

The dosage of the pharmaceutical composition can be determined readilyby the skilled artisan, for example, by first identifying doseseffective to elicit a prophylactic or therapeutic immune response, bymeasuring the serum titer of virus specific immunoglobulins or bymeasuring the inhibitory ratio of antibodies in serum samples, or urinesamples, or mucosal secretions. The dosages can be determined fromanimal studies. A non-limiting list of animals used to study theefficacy of vaccines include the guinea pig, hamster, ferrets,chinchilla, mouse and cotton rat. Most animals are not natural hosts toinfectious agents but can still serve in studies of various aspects ofthe disease. For example, any of the above animals can be dosed with avaccine candidate, e.g. modified or mutated RSV F proteins, an RSV Fmicelle comprising a modified or mutated RSV F protein, or VLPs of theinvention, to partially characterize the immune response induced, and/orto determine if any neutralizing antibodies have been produced. Forexample, many studies have been conducted in the mouse model becausemice are small size and their low cost allows researchers to conductstudies on a larger scale.

In addition, human clinical studies can be performed to determine thepreferred effective dose for humans by a skilled artisan. Such clinicalstudies are routine and well known in the art. The precise dose to beemployed will also depend on the route of administration. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal test systems.

As also well known in the art, the immunogenicity of a particularcomposition can be enhanced by the use of non-specific stimulators ofthe immune response, known as adjuvants. Adjuvants have been usedexperimentally to promote a generalized increase in immunity againstunknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocolshave used adjuvants to stimulate responses for many years, and as such,adjuvants are well known to one of ordinary skill in the art. Someadjuvants affect the way in which antigens are presented. For example,the immune response is increased when protein antigens are precipitatedby alum. Emulsification of antigens also prolongs the duration ofantigen presentation. The inclusion of any adjuvant described in Vogelet al., “A Compendium of Vaccine Adjuvants and Excipients (2^(nd)Edition),” herein incorporated by reference in its entirety for allpurposes, is envisioned within the scope of this invention.

Exemplary, adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant. Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDPcompounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, andmonophosphoryl lipid A (MPL). RIBI, which contains three componentsextracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wallskeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated.MF-59, Novasomes®, MHC antigens may also be used.

In one embodiment of the invention the adjuvant is a paucilamellar lipidvesicle having about two to ten bilayers arranged in the form ofsubstantially spherical shells separated by aqueous layers surrounding alarge amorphous central cavity free of lipid bilayers. Paucilamellarlipid vesicles may act to stimulate the immune response several ways, asnon-specific stimulators, as carriers for the antigen, as carriers ofadditional adjuvants, and combinations thereof. Paucilamellar lipidvesicles act as non-specific immune stimulators when, for example, avaccine is prepared by intermixing the antigen with the preformedvesicles such that the antigen remains extracellular to the vesicles. Byencapsulating an antigen within the central cavity of the vesicle, thevesicle acts both as an immune stimulator and a carrier for the antigen.In another embodiment, the vesicles are primarily made ofnonphospholipid vesicles. In other embodiment, the vesicles areNovasomes®. Novasotnes® are paucilamellar nonphospholipid vesiclesranging from about 100 nm to about 500 nm. They comprise Brij 72,cholesterol, oleic acid and squalene. Novasomes have been shown to be aneffective adjuvant for influenza antigens (see, U.S. Pat. Nos.5,629,021, 6,387,373, and 4,911,928, herein incorporated by reference intheir entireties for all purposes).

The compositions of the invention can also be formulated with “immunestimulators.” These are the body's own chemical messengers (cytokines)to increase the immune system's response. Immune stimulators include,but not limited to, various cytokines, lymphokines and chemokines withimmunostimulatory, immunopotentiating, and pro-inflammatory activities,such as interleukins (e.g., IL1, IL-2, IL-3, IL-4, IL-12, IL-13); growthfactors (e.g., granulocyte-macrophage (GM)-colony stimulating factor(CSF)); and other immunostimulatory molecules, such as macrophageinflammatory factor, Flt3 ligand, B7.1; B7,2, etc. The immunostimulatorymolecules can be administered in the same formulation as thecompositions of the invention, or can be administered separately. Eitherthe protein or an expression vector encoding the protein can beadministered to produce an immunostimulatory effect. Thus in oneembodiment, the invention comprises antigentic and vaccine formulationscomprising an adjuvant and/or an immune stimulator.

Methods of Stimulating an Immune Response

The modified or mutated. RSV F proteins, the RSV F micelles comprising amodified or mutated RSV F protein, or the VLPs of the invention areuseful for preparing compositions that stimulate an immune response thatconfers immunity or substantial immunity to infectious agents. Bothmucosal and cellular immunity may contribute to immunity to infectiousagents and disease. Antibodies secreted locally in the upperrespirators, tract are a major factor in resistance to naturalinfection. Secretory immunoglobulin A (slgA) is involved in theprotection of the upper respiratory tract and serum IgG in protection ofthe lower respiratory tract. The immune response induced by an infectionprotects against reinfection with the same virus or an antigenicallysimilar viral strain. For example, RSV undergoes frequent andunpredictable changes; therefore, after natural infection, the effectiveperiod of protection provided by the host's immunity may only beeffective for a few years against the new strains of virus circulatingin the community.

Thus, the invention encompasses a method of inducing immunity toinfections or at least one disease symptom thereof in a subject,comprising administering at least one effective dose of a modified ormutated RSV F protein, an RSV F micelle comprising a modified or mutatedRSV F protein, or a VLP comprising a modified or mutated RSV F protein.In one embodiment, the method comprises administering VLPs comprising amodified or mutated RSV F protein and at least one additional protein.In another embodiment, the method comprises administering VLPs furthercomprising an RSV M protein, for example, a BRSV M protein. In anotherembodiment, the method comprises administering VLPs further comprising aRSV N protein. In another embodiment, the method comprises administeringVLPs further comprising a RSV G protein. In another embodiment, themethod comprises administering VLPs further comprising a RSV SH protein.In another embodiment, the method comprises administering VLPs furthercomprising F and/or G protein from HRSV group A and/or group B. Inanother embodiment, the method comprises administering VLPs comprising Mprotein from BRSV and a chimeric RSV F and/or G protein or MMTV envelopeprotein, for example, HA or NA protein derived from an influenza virus,wherein the HA and/or NA protein is fused to the transmembrane domainand cytoplasmic tail of the RSV F and/or G protein or MMTV envelopeprotein. In another embodiment, the method comprises administering VLPscomprising M protein from BRSV and a chimeric RSV F and/or G protein andoptionally HA or NA protein derived from an influenza virus, wherein theHA or NA protein is fused to the transmembrane domain and cytoplasmictail of RSV F and/or G protein. In another embodiment, the subject is amammal. In another embodiment, the mammal is a human. In anotherembodiment, RSV VLPs are formulated with an adjuvant or immunestimulator.

In one embodiment, the invention comprises a method to induce immunityto RSV infection or at least one disease symptom thereof in a subject,comprising administering at least one effective dose of a modified ormutated RSV F protein, In another embodiment, the invention comprises amethod to induce immunity to RSV infection or at least one diseasesymptom thereof in a subject, comprising administering at least oneeffective dose of an RSV F micelle comprising a modified or mutated RSVF protein. In yet another embodiment, the invention comprises a methodto induce immunity to RSV infection or at least one disease symptomthereof in a subject, comprising administering at least one effectivedose of a RSV VLPs, wherein the VLPs comprise a modified or mutated RSVF protein, M, G, SH, and/or N proteins. In another embodiment, a methodof inducing immunity to RSV infection or at least one symptom thereof ina subject, comprises administering at least one effective dose of a RSVVLPs, wherein the VIPs consists essentially of BRSV M (includingchimeric M), and RSV F, G, and/or or N proteins. The VLPs may compriseadditional RSV proteins and/or protein contaminates in negligibleconcentrations. In another embodiment, a method of inducing immunity toRSV infection or at least one symptom thereof in a subject, comprisesadministering at least one effective dose of a RSV VLPs, wherein theVLPs consists of BRSV M (including chimeric M), RSV G and/or F. Inanother embodiment, a, method of inducing immunity to RSV infection orat least one disease symptom in a subject, comprises administering atleast one effective dose of a RSV VLPs comprising RSV proteins, whereinthe RSV proteins consist of BRSV M (including chimeric M), F, G, and/orN proteins, including chimeric F, G, and/or or N proteins. These VLPscontain BRSV M (including chimeric M), RSV F, G, and/or N proteins andmay contain additional cellular constituents such as cellular proteins,baculovirus proteins, lipids, carbohydrates etc., but do not containadditional RSV proteins (other than fragments of BRSV M (includingchimeric M), BRSV/RSV F, G, and/or N proteins. In another embodiment,the subject is a vertebrate. In one embodiment the vertebrate is amammal. In another embodiment, the mammal is a human. In anotherembodiment, the method comprises inducing immunity to RSV infection orat least one disease symptom by administering the formulation in onedose. In another embodiment, the method comprises inducing immunity toRSV infection or at least one disease symptom by administering theformulation in multiple doses.

The invention also encompasses inducing immunity to an infection, or atleast one symptom thereof, in a subject caused by an infectious agent,comprising administering at least one effective dose of a modified ormutated RSV F protein, an RSV F micelle comprising a modified or mutatedRSV F protein, or a VLP comprising a modified or mutated RSV F protein.In one embodiment, the method comprises administering VLPs comprising amodified or mutated RSV F protein and at least one protein from aheterologous infectious agent. In one embodiment, the method comprisesadministering VLPs comprising a modified or mutated RSV F protein and atleast one protein from the same or a heterologous infectious agent. Inanother embodiment, the protein from the heterologous infectious agentis a viral protein. In another embodiment, the protein from theinfectious agent is an envelope associated protein. In anotherembodiment, the protein from the infectious agent is expressed on thesurface of VLPs. In another embodiment, the protein from the infectiousagent comprises an epitope that will generate a protective immuneresponse in a vertebrate. In another embodiment, the protein from theinfectious agent can associate with RSV M protein such as BRSV Mprotein, RSV F, G and/or N protein. In another embodiment, the proteinfrom the infectious agent is fused to a RSV protein such as a BRSV Mprotein, RSV F, G and/or N protein, in another embodiment, only aportion of a protein from the infectious agent is fused to a RSV proteinsuch as a BRSV M protein, RSV F, G and/or N protein. In anotherembodiment, only a portion of a protein from the infectious agent isfused to a portion of a RSV protein such as a BRSV M protein, RSV F, Gand/or N protein. In another embodiment, the portion of the protein fromthe infectious agent fused to the RSV protein is expressed on thesurface of VLPs. In other embodiment, the RSV protein, or portionthereof, fused to the protein from the infectious agent associates withthe RSV M protein. In other embodiment, the RSV protein, or portionthereof, is derived from RSV F, G, N and/or P. In another embodiment,the chimeric VLPs further comprise N and/or P protein from RSV. Inanother embodiment, the chimeric VLPs comprise more than one proteinfrom the same and/or a heterologous infectious agent. In anotherembodiment, the chimeric. VLPs comprise more than one infectious agentprotein, thus creating a multivalent VLP.

Compositions of the invention can induce substantial immunity in avertebrate (e.g. a human) when administered to the vertebrate. Thesubstantial immunity results from an immune response againstcompositions of the invention that protects or ameliorates infection orat least reduces a symptom of infection in the vertebrate. In someinstances, if the vertebrate is infected, the infection will beasymptomatic. The response may not be a fully protective response. Inthis case, if the vertebrate is infected with an infectious agent, thevertebrate will experience reduced symptoms or a shorter duration ofsymptoms compared to a non-immunized vertebrate.

In one embodiment, the invention comprises a method of inducingsubstantial immunity to RSV virus infection or at least one diseasesymptom in a subject, comprising administering at least one effectivedose of a modified or mutated RSV F protein, an RSV F micelle comprisinga modified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein. In another embodiment, the invention comprises amethod of vaccinating a mammal against RSV comprising administering tothe mammal a protection-inducing amount of a modified or mutated RSV Fprotein, an RSV F micelle comprising a modified or mutated RSV Fprotein, or a VLP comprising a modified or mutated RSV F protein. In oneembodiment, the method comprises administering VLPs further comprisingan RSV M protein, such as BRSV M protein. In another embodiment, themethod further comprises administering VLPs comprising RSV G protein,for example a HRSV G protein. In another embodiment, the method furthercomprises administering VLPs comprising the N protein from HRSV group A.In another embodiment, the method further comprises administering VLPscomprising the N protein from HRSV group B. In another embodiment, themethod comprises administering VLPs comprising chimeric M protein fromBRSV and F and/or G protein derived from RSV wherein the F and/or Gprotein is fused to the transmembrane and cytoplasmic tail of the Mprotein. In another embodiment, the method comprises administering VLPscomprising M protein from BRSV and chimeric RSV F and/or G proteinwherein the F and/or G protein is a fused to the transmembrane domainand cytoplasmic tail of influenza HA and/or NA protein. In anotherembodiment, the method comprises administering VLPs comprising M proteinfrom BRSV and chimeric RSV F and/or G protein and optionally aninfluenza HA and/or NA protein wherein the F and/or G protein is a fusedto the transmembrane domain and cytoplasmic tail of the HA protein. Inanother embodiment, the method comprises administering VLPs comprising Mprotein from BRS and chimeric RSV F and/or G protein, and optionally aninfluenza. HA and/or NA protein wherein the HA and/or NA protein isfused to the transmembrane domain and cytoplasmic tail of RSV F and/or Gprotein.

The invention also encompasses a method of inducing substantial immunityto an infection, or at least one disease symptom in a subject caused byan infectious agent, comprising administering at least one effectivedose of a modified or mutated RSV F protein, an RSV F micelle comprisinga modified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein. In one embodiment, the method comprisesadministering VLPs further comprising a RSV M protein, such as BRSV Mprotein, and at least one protein from another infectious agent. In oneembodiment, the method comprises administering VLPs further comprising aBRSV M protein and at least one protein from the same or a heterologousinfectious agent. In another embodiment, the protein from the infectiousagent is a viral protein. In another embodiment, the protein from theinfectious agent is an envelope associated protein. In anotherembodiment, the protein from the infectious agent is expressed on thesurface of VLPs. In another embodiment, the protein from the infectiousagent comprises an epitope that will generate a protective immuneresponse in a vertebrate. In another embodiment, the protein from theinfectious agent can associate with RSV M protein. In anotherembodiment, the protein from the infectious agent can associate withBRSV M protein. In another embodiment, the protein from the infectiousagent is fused to a RSV protein. In another embodiment, only a portionof a protein from the infectious agent is fused to a RSV protein. Inanother embodiment, only a portion of a protein from the infectiousagent is fused to a portion of a RSV protein. In another embodiment, theportion of the protein from the infectious agent fused to the RSVprotein is expressed on the surface of VLPs. In other embodiment, theRSV protein, or portion thereof, fused to the protein from theinfectious agent associates with the RSV M protein. In other embodiment,the RSV protein, or portion thereof, fused to the protein from theinfectious agent associates with the BRSV M protein. In otherembodiment, the RSV protein, or portion thereof, is derived from RSV F,G, N and/or P. In another embodiment, the VLPs further comprise N and/orP protein from RSV. In another embodiment, the VLPs comprise more thanone protein from the infectious agent. In another embodiment, the VLPscomprise more than one infectious agent protein, thus creating amultivalent VLP.

In another embodiment, the invention comprises a method of inducing aprotective antibody response to an infection or at least one symptomthereof in a subject, comprising administering at least one effectivedose of a modified or mutated RSV F protein, an RSV F micelle comprisinga modified or mutated RSV F protein, or a VLP comprising a modified ormutated RSV F protein as described above.

As used herein, an “antibody” is a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which intum define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. Antibodies exist as intactimmunoglobulins or as a number of well-characterized fragments producedby digestion with various peptidases.

In one embodiment, the invention comprises a method of inducing aprotective cellular response to RSV infection or at least one diseasesymptom in a subject, comprising administering at least one effectivedose of a modified or mutated RSV F protein. In another embodiment, theinvention comprises a method of inducing a protective cellular responseto RSV infection or at least one disease symptom in a subject,comprising administering at least one effective dose an RSV F micellecomprising a modified or mutated RSV F protein. In yet anotherembodiment, the invention comprises a method of inducing a protectivecellular response to RSV infection or at least one disease symptom in asubject, comprising administering at least one effective dose a VLP,wherein the VLP comprises a modified or mutated RSV F protein asdescribed above. Cell-mediated immunity also plays a role in recoveryfrom RSV infection and may prevent RSV-associated complications.RSV-specific cellular lymphocytes have been detected in the blood andthe lower respiratory tract secretions of infected subjects. Cytolysisof RSV-infected cells is mediated by CTLs in concert with RSV-specificantibodies and complement. The primary cytotoxic response is detectablein blood after 6-14 days and disappears by day 21 in infected orvaccinated individuals (Ennis et al., 1981). Cell-mediated immunity mayalso play a role in recovery from RSV infection and may preventRSV-associated complications. RSV-specific cellular lymphocytes havebeen detected in the blood and the lower respiratory tract secretions ofinfected subjects.

As mentioned above, the immunogenic compositions of the inventionprevent or reduce at least one symptom of RSV infection in a subject.Symptoms of RSV are well known in the art. They include rhinorrhea, sorethroat, headache, hoarseness, cough, sputum, fever, rales, wheezing, anddyspnea. Thus, the method of the invention comprises the prevention orreduction of at least one symptom associated with RSV infection. Areduction in a symptom may be determined subjectively or objectively,e.g., self assessment by a subject, by a clinician's assessment or byconducting an appropriate assay or measurement (e.g. body temperature),including, e.g., a quality of life assessment, a slowed progression of aRSV infection or additional symptoms, a reduced severity of a RSVsymptoms or a suitable assays (e.g. antibody titer and/or T-cellactivation assay). The objective assessment comprises both animal andhuman assessments.

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference for all purposes.

EXAMPLES Example 1 Generating Recombinant Bacmids, Transfection ofInsect Cells To Make Recombinant Virus Stocks, Plaque Purification, andInfecting Insect Cells with Primary Virus Stock

To construct recombinant virus, the viral genes of interest were codonoptimized for Sf9 insect cells expression and cloned into pFastBac™vectors.

Once the desired constructs were confirmed and purified, one vial of MAXEfficiency® DH10Bac™ competent cells for each construct was thawed onice. Approximately 1 ng (5 μl) of the desired pFastBac™ constructplasmid DNA was added to the cells and mixed gently. The cells wereincubated on ice for 30 minutes. This was followed by heat-shock of thecells for 45 seconds at 42° C. without shaking. Next, the tubes weretransferred to ice and chilled for 2 minutes. Subsequently 900 μl ofroom temperature S.O.C. Medium was added to each tube. The tubes wereput on a shaker at 37° C. at 225 rpm for 4 hours. For each pFastBc™transformation, 10-fold serial dilutions of the cells (10-1, 10-2 and10-3) was prepared using S.O.C. medium. Next, 100 μl of each dilutionwas plated on an LB agar plate containing 50 μg/ml kanamycin, 7 μg/mlgentamicin, 10 μg/ml tetracycline, 100 μg/ml Bluo-gal, and 40 μg/mlIPTG. The plates were incubated for 48 hours at 37° C. White colonieswere picked for analysis.

Different bacmid DNAs from above were made for each construct and wereisolated. These DNAs were precipitation and added to Sf9 cells for 5hours.

Next, 30 ml of Sf9 insect cells (2×10⁶ cells/ml) were infected withbaculovirus expressing viral proteins of interest with 0.3 ml of plaqueeluate and incubated 48-72 hrs. Approximately 1 ml of crude culture(cells+medium) and clarified culture harvests were saved for expressionanalysis and the rest were saved for purification purposes.

Example 2 Expression, Purification, and Analysis of Modified HRSV FProteins

Genes encoding modified HRSV F proteins of interest were synthesized invitro as overlapping oligonucleotides, cloned and expressed in hostcells. Cloning and expression of the modified RSV F genes were achievedfollowing the methods known in the art.

Recombinant plaques containing viral proteins of interest were pickedand confirmed. The recombinant virus was then amplified by infection ofSf9 insect cells. In some cases, Sf9 insect cells were co-infected by arecombinant virus expressing modified F protein and another recombinantvirus expressing other viral proteins (e.g., BRSV M protein and/or HRSVN protein). A culture of insect cells was infected at ˜3 MOI(Multiplicity of infection=virus ffu or pfu/cell) with baculoviruscarrying the various constructs. The culture and supernatant wereharvested 48-72 post-infection. The crude harvest, approximately 30 mL,was clarified by centrifugation for 15 minutes at approximately 800×g.The resulting crude cell harvests containing modified HRSV F proteinwere purified as described below.

Modified HRSV F proteins of interest were purified from the infected Sf9insect cell culture harvests. Non-ionic surfactant Tergitol® NP-9(Nonylphenol Ethoxylate) was used to in a membrane protein extractionprotocol. Crude extraction was further purified by passing through anionexchange chromatography, lentil lectin affinity/HIC, and cation exchangechromatography.

Protein expression was analyzed by SDS-PAGE and stained for totalproteins by coomassie stain. Equal volumes of cell samples from crudeharvest and 2× sample buffer containing βME (beta-mercaptoehtanol) wereloaded, approximately 15 to 20 μl (about to 7.5 to 10 μl of theculture)/lane, onto an SDS Laemmli gel.

In some cases, instead of chromatography, modified. HRSV F proteins inthe crude cell harvests were concentrated by 30% sucrose gradientseparation method, and then were analyzed by SDS-PAGE stained withcoomassie, or Western Blot using anti-RSV F monoclonal antibody.

Crude cell harvest containing modified recombinant F proteins, purifiedrecombinant F proteins, or recombinant F proteins concentrated bysucrose gradient can be further analyzed by Western Blot using anti-RSVF monoclonal antibody and/or anti-RSV F polyclonal antibody.

Example 3 Modified HRSV F Gene Encoding F Protein BV #541

Initial attempts to express the full length HRSV F protein provedunsuccessful in achieving high levels of expression, The F gene sequenceused in the expression was SEQ ID NO: 1 (wild type HRSV F gene, GenBankAccession No. M11486). It encodes an inactive precursor (F₀) of 574 aa.This precursor is cleaved twice by furin-like proteases duringmaturation to yield two disulfide-linked polypeptides, subunit F₂ fromthe N terminus and F₁ from the C terminus (FIG. 1 ). The two cleavagessites are at residues 109 and 136, which are preceded byfurin-recognition motifs (RARR, aa 106-109 (SEQ ID NO: 23) and KKRKRR,aa 131-136 (SEQ ID NO: 24)). The F gene sequence of SEQ ID NO: 1contains suboptimal codon usage for expression in SP9 insect cells andharbors 3 errors, producing a protein that can exhibit less than optimalfolding (SEQ ID NO: 2, GenBank Accession No. AAB59858). In addition, apossible Poly (A) adenylation site (ATAAAA) was identified at the regionencoding the F₂ subunit. Moreover, the wild type F gene sequence isapproximately 65% AT rich, while desired GC-AT ratio of a gene sequencein Sf9 insect cell expression system is approximately 1:1.

In attempt to overcome poor expression levels of HRSV F protein, a new Fgene sequence was designed so that:

(a) the three GenBank sequencing errors were corrected;

(b) the cryptic poly (A) site at the region encoding F2 subunit wasmodified;

(c) F gene codons were optimized; and

(d) the F gene encodes a modified F protein with inactivated primarycleavage site.

The three corrected amino acids errors were P102A, I379V, and M447V. Thecryptic poly (A) site in the HRSV F gene was corrected without changingthe amino acid sequence.

The codon optimization scheme was based on the following criteria: (1)abundance of aminoacyl-tRNAs for a particular codon in Lepidopteranspecies of insect cells for a given amino acid as described by Levin, D.B. et al, (Journal of General Virology, 2000, vol. 81, pp. 2313-2325),(2) maintenance of GC-AT ratio in gene sequences at approximately 1:1,(3) minimal introduction of palindromic or stem-loop DNA structures, and(4) minimal introduction of transcription and post-transcriptionrepressor element sequences. An example of optimized F gene sequence wasshown as SEQ ID NO: 19 (RSV-F BV #368).

To inactivate the primary cleavage site (1° CS. KKRKRR, aa 131-136) ofHRSV F protein, the furin recognition site was mutated to either KKQKQQ(SEQ ID NO: 28) or GRRQQR (SEQ ID NO: 29). Several modified F proteinswith such cleavage site mutations were evaluated to determine theefficiency of cleavage prevention. FIG. 2 shows several of the modifiedF proteins that were evaluated. The results indicate that the primarycleavage site of HRSV F protein can be inactivated by three conservativeamino acid changes R133Q, R135Q, and R136Q. These conservative aminoacid changes from Arginine (R) which is a polar-charged molecule, toGlutamine (Q) which is a polar-neutral molecule, altered the chargestatus at these sites and prevented cleavage by furin-like proteases(see FIG. 3 ), while still preserving the F protein 3D structure.Prevention of cleavage at 1° CS resulted reduced membrane fusionactivity of the F protein.

A non-limiting exemplary modified HRSV F gene sequence designed to haveall modifications mentioned above is shown in FIG. 4 . This modified Fgene (SEQ ID NO: 5, RSV-F BV #541) encodes a modified F protein of SEQID NO: 6. The gene sequence was synthesized in vitro as overlappingoligonucleotides, cloned and expressed in host cells. Modified HRSV Fprotein BV #541 was purified from the infected Sf9 insect cell cultureharvests, and was analyzed by SDS-PAGE stained by coomassie. The methodof purification and SDS-PAGE analysis is described in Example 2. Theexpression level of the F protein RSV-F BV #541 (e.g. F protein 541 wasimproved as compared to the wild type F₀ protein in SF9 insect cells.

Example 4 Modified HRSV F Protein with F₁ Subunit Fusion Domain PartialDeletion

To further improve expression of the RSV F protein, additionallymodified HRSV F genes were designed that comprised the followingmodifications:

(a) the three GenBank sequencing errors were corrected;

(b) the cryptic poly (A) site at the region encoding F2 subunit wasmodified;

(c) F gene codons were optimized; and

(d) the nucleotide sequences encoding the F₁ subunit fusion domain waspartially deleted. In one experiment, the nucleotide sequence encodingthe first 10 amino acids of the F₁ subunit fusion domain was deleted(corresponding to amino acids 137-146 of SEQ ID NO: 2).

A non-limiting exemplary modified. RSV F gene comprising saidmodifications is shown in FIG. 5 , designated as SEQ ID NO: 9 (RSV-F BV#622, e.g. F protein 622), encoding a modified F protein of SEQ ID NO:10. The modified HRSV F protein BV #622 was purified from the infectedSF9 insect cell culture harvests, and was analyzed by SDS-PAGE stainedwith coomassie. The method of purification and SDS-PAGE analysis isdescribed in Example 2. High expression levels of HRSV F protein BV #622were observed, as displayed in the SDS-PAGE FIG. 6 .

Example 5 Modified HRSV F Protein with Both Inactivated Primary CleavageSite and F₁ Fusion Domain Partial Deletion

To determine if the combination of inactivated primary cleavage site andF₁ fusion domain partial deletion can further promote expression of theRSV F protein, particularly in the Sf9 insect cells, another modifiedRSV F gene was designed comprising following modifications:

(a) the three GenBank sequencing errors were corrected;

(b) the cryptic poly (A) site at the region encoding F2 subunit wasmodified;

(c) F gene codons were optimized;

(d) the primary cleavage site was inactivated; and

(e) the nucleotide sequence encoding the F1 subunit fusion domain waspartially deleted. In one experiment, the nucleotide sequence encodingthe first 10 amino acids of the F₁ subunit fusion domain was deleted(corresponding to amino acids 137-146 of SEQ ID NO: 2).

An non-limiting exemplary modified RSV F gene comprising saidmodifications is shown in FIG. 7 , designated as SEQ ID NO: 7 (RSV-F BV#683, e.g. F protein 683), encoding the modified F protein of SEQ ID NO:8. The modified RSV F protein BV #683 (e.g. F protein 683) was purifiedfrom the infected Sf9 insect cell culture harvests and analyzed bySDS-PAGE stained with coomassie. The method of purification and SDS-PAGEanalysis is described in Example 2. Further enhancements in the ofexpression levels were achieved, as displayed in the SDS-PAGE in FIG. 8.

Example 6 Expression and Purification of Modified HRSV F Protein BV #683

Modified HRSV F protein BV #683 (e.g. F protein 683, SEQ ID NO: 8) wasexpressed in baculovirus expression system as describe in Example 1, andrecombinant plaques expressing HRSV F protein BV #683 were picked andconfirmed. The recombinant virus was then amplified by infection of Sf9insect cells. A culture of insect cells was infected at ˜3 MOI(Multiplicity of infection=virus ffu or pfu/cell) with baculovirus. Theculture and supernatant were harvested 48-72 hrs post-infection. Thecrude harvest, approximately 30 mL, was clarified by centrifugation for15 minutes at approximately 800×g. The resulting crude cell harvestscontaining HRSV F protein BV #683 were purified as described below.

HRSV F protein BV #683 was purified from the infected Sf9 insect cellculture harvests. Non-ionic surfactant Tergitol® NP-9 (NonylphenolEthoxylate) was used to in a membrane protein extraction protocol. Crudeextraction was further purified by passing through anion exchangechromatography, lentil lectin affinity/HIC and cation exchangechromatography.

Purified HRSV F protein BV #683 was analyzed by SDS-PAGE stained withcoomassie, and Western Blot using anti-RSV F monoclonal antibody asdescribed in Example 2. The results were shown in FIG. 9 . Excellentexpression levels of the HRSV F protein BV #683 (e.g. F protein 683, SEQID NO: 8) were achieved. It was estimated that the expression level wasabove 10 mg/L in crude cell culture, and recovered F protein BV #683 wasabout 3.5 mg/L cell culture. In some cases expression levels above 20mg/L were achieved and about 5 mg/L modified F protein BV #683 wasrecovered (see FIG. 10 ). Purity of the recovered F protein BV #683reached above 98% as determined by scanning densitometry (see FIG. 10 ).

Example 7 Purified HRSV F Protein BV #683 Micelles (Rosettes)

Purified HRSV F protein BV #683 was analyzed by negative stain electronmicroscopy (see FIG. 11 ). F proteins aggregated in the form of micelles(rosettes), similar to those observed for wild type HRSV F protein(Calder et al., 2000, Virology 271, pp. 122-131), and other full-lengthvirus membrane glycoproteins (Wrigley et al., Academic Press, London,1986. vol. 5, pp. 103-163). Under electron microscopy, the F spikesexhibited lollipop-shaped rod morphology with their wider endsprojecting away from the centers of the rosettes. The length of singletrimer was about 20 nm, and the micelle particle diameter was about 40nm (see FIG. 12 ). These results indicated that HRSV F protein BV #683has correct 3D structure for a native, active protein.

In summary, a modified recombinant HRSV F protein (e.g., BV #683) hasbeen designed, expressed, and purified. This modified full-length F isglycosylated. Modifications of the primary cleavage site and the fusiondomain together greatly enhanced expression level of F protein. Inaddition, this modified F protein can be cleaved to F₁ and F₂ subunits,which are disulfide-linked. Trimers of the F₁ and F₂ subunits formloilipop-shaped spikes of 19.6 nm and particles of 40.2 nm. Moreover,this modified F protein is highly expressed in Sf9 insect cells. Purityof micelles >98% is achieved after purification. The fact that thespikes of this modified protein have a lollipop morphology, which canfurther form micelles particles of 40 nm, indicates that modified Fprotein BV #683 has correct 3D structure of a native protein.

Example 8 Co-Expression of Modified HRSV F Protein with BRSV M and/orHRSV N in VLP Production

The present invention also provides VLPs comprising a modified ormutated RSV F protein. Such VLPs are useful to induce neutralizingantibodies to viral protein antigens and thus can be administered toestablish immunity against RSV. For example, such VLPs may comprise amodified RSV F protein, and a BRSV M and/or HRSV N proteins. Codons ofgenes encoding BRSV M (SEQ ID NO: 14) or HRSV N (SEQ ID NO: 18) proteinscan be optimized for expression in insect cells. For example, anoptimized BRSV M gene sequence is shown in SEQ ID NO: 13 and anoptimized RSV N gene sequence is shown in SEQ ID NO: 17.

In one experiment, a modified F protein BV #622 and another modified Fprotein BV #623 (SEQ ID NO: 21, modified such that both cleavage sitesare inactivated) were either expressed alone, or co-expressed with HRSVN protein and BRSV M protein. Both crude cell harvests containing VLPs(intracellular) and VLPs pellets collected from 30% sucrose gradientseparation were analyzed by SDS-PAGE stained with coomassie, and WesternBlot using anti-RSV F monoclonal antibody. FIG. 13 shows the structureof the modified F proteins BV #622 and BV #623, and results of SDS-PAGEand Western Blot analysis. BV #622 was highly expressed by itself orco-expressed with HRSV N protein and BRSV M protein, while BV #623 hadvery poor expression, indicating inactivation of both cleavage sitesinhibits F protein expression.

In another experiment, modified F protein BV #622, double tandem gene BV#636 (BV #541+BRSV M), BV #683, BV #684 (BV #541 with YIAL L-domainintroduced at the C terminus), and BV #685 (BV #541 with YKKL L-domainintroduced at the C terminus) were either expressed alone, orco-expressed with HRSV N protein and BRSV M protein. L-domain (Latedomain) is conserved sequence in retroviruses, and presents within Gagacting in conjunction with cellular proteins to efficiently releasevirions from the surface of the cell (Ott et al., 2005, Journal ofVirology 79: 9038-9045). The structure of each modified F protein isshown in FIG. 14 . Both crude cell harvests containing VLPs(intracellular) and VLPs pellets collected from 30% sucrose gradientseparation were analyzed by SDS-PAGE stained with coomassie, and WesternBlot using anti-RSV F monoclonal antibody. FIG. 14 shows the results ofSDS-PAGE and Western Blot analysis of crude cell harvests containingVLPs (intracellular), and FIG. 15 showed results of SDS-PAGE and WesternBlot analysis of VLPs pellets collected from 30% sucrose gradientseparation. BV #622 and BV #683 were highly expressed by themselves orco-expressed with HRSV N protein and BRSV M protein, while BV #636, BV#684, and BV #685 had poor expression.

Example 9 Screening of Chimeric HRSV F Proteins with High Expression

Efforts were made to screen for additional RSV F proteins that can behighly expressed in soluble form in insect cells and can form VLPs withbetter yield. Various F genes were designed, expressed, and analyzed.Both Western Blot and SDS-PAGE were used to evaluate the expression.

FIG. 16 a to FIG. 16 d summarize the structure, clone name, description,Western Bloticoomassie analysis results, and conclusion for eachchimeric HRSV F clone.

As the results indicated, wild type full length F protein was poorlyexpressed; chimeric HRSV F proteins that contain F₁ but not F₂ subunitcould be expressed well, but the products were either insoluble, whichmight be due to misfolding, or could not assemble with other viralproteins to form VLPs with good yield after co-infections. Inactivationof the primary cleavage site alone did not result in substantialincreases in expression, but better expression was achieved wheninactivation of the primary cleavage site was combined with othermodification such as deletion of cryptic poly (A) site and correction ofGenBank aa errors e.g., BV #541). Introduction of the YKKL L-domain intothe C terminus of BV #541 enhanced the secretion of VLPs containingmodified F protein for about 2-3 folds in co-expression with BRSV M andHRSV N proteins. The results further showed that a double tandemchimeric gene consisting of BV #541 gene and BRSV M gene displayed bothimproved intracellular and VLPs yield compared to co-infection of BV#541 and BRSV M proteins, indicating that BRSV M protein can facilitateproduction of VLPs containing modified HRSV F protein in insect cellswhen tandemly expressed. A triple tandem chimeric gene consisting of BV#541, BRSV M, and HRSV N had even higher intracellular and much betterVLPs yield compared to above mentioned double tandem chimeric gene orco-infection of BV #541, BRSV M, and HRSV N proteins. Furthermore; theresults suggested that chimeric HRSV F protein BV#683 (e.g. F protein683, SEQ ID NO: 8) had the best intracellular expression. Expression ofa double tandem chimeric gene consisting of BV#683 and BRSV M genes, ora triple tandem chimeric gene consisting of BV#683, BRSV M, and HRSV Ngenes is also embodied herein. These double and triple tandem chimericgene should further improve VLP production compared to co-infection.

Example 10 RSV Neutralization Assay and RSV Challenge Studies in Mice

To test the efficiency of vaccine comprising modified HRSV F protein BV#683 in prohibiting RSV infection, neutralization assay and RSVchallenge studies were conducted in mice. The experimental proceduresare shown in FIG. 17 .

Groups of mice (n=10) were injected intramuscularly (except for liveRSV) with placebo (PBS solution), live RSV (given intranasally),formalin inactivated RSV vaccine (FI-RSV), 1 ug purified F particles(PFP, modified F protein BV #683), 1 ug purified F particles with Alum(PFP+Alum), 10 ug purified F particles, 10 ug purified F particles withAlum (PFP+Alum), or 30 ug purified F particles on day 0 and day 21. Eachimmunized group was challenged by live RSV on day 42 (21 days after thesecond immunization). Mouse serum from each group was harvested on day0, day 31 (10 days after the second immunization), and day 46 (4 daysfollowing challenge with live RSV).

Mouse serum from each treatment group was assayed for the presence ofanti-RSV neutralization antibodies. Dilutions of serum from immunizedmice were incubated with infectious RSV in 96-well microtiter plates.Serum was diluted from 1:20 to 1:2560. 50 ul diluted serum was mixedwith 50 ul live RSV virus (400 pfu) in each well. The virus/serummixture was incubated first for 60 minutes at room temperature, and thenmixed with 100 ul HEp-2 cells and incubated for 4 days. The number ofinfectious virus plaques were then counted after stained with crystalviolet. The neutralization titer for each serum sample was defined asthe inverse of the highest dilution of serum that produced 100% RSVneutralization (e.g., no plaques) and was determined for each animal.The geometric mean serum neutralizing antibody titer at day 31 (10 daysafter the boost) and day 46 (4 days following challenge with live RSV)were graphed for each vaccine group. FIG. 18 shows the results ofneutralization assays. The results indicate that 10 ug or 30 ug purifiedF protein produce much higher neutralization titer as compared to liveRSV. In addition, neutralization titers of PEP are enhanced withco-administration of Alum adjuvant,

RSV challenge studies were carried out to determine if immunizationcould prevent and/or inhibit RSV replication in the lungs of theimmunized animals. The amount of RSV in the lungs of immunized mice wasdetermined by plaque assay using HEp-2 cells. Immunized groups of micementioned above were infected with 1×10⁶ pfu of infectious RSV longstrain intranasally on day 42 (11 days after the second immunization).On day 46 (4 days after RSV infection), lungs of mice were removed,weighed, and homogenized. Homogenized lung tissue was clarified.Supernatant of clarified solution was diluted and subjected to plaqueassay using HEp-2 cells to determine RSV titer in lung tissue(calculated as pfu/g lung tissue). Results are shown in FIG. 19 ,indicating that all mice immunized with recombinant RSV F protein By#683 had undetectable RSV in the lungs, and even 1 ug purifiedrecombinant HRSV F protein BV #683 without adjuvant exhibited excellentefficiency in inhibiting RSV replication (reduced more then 1000 timescompared to placebo).

To determine the stability of RSV PIT vaccine used above, the vaccinewas stored at 2-8° C. for 0, 1, 2. 4, and 5 weeks, and then analyzed bySDS-PAGE stained with coomassie (FIG. 20 ). The results show that thisRSV PFP vaccine is very stable at 2-8° C. and there is no detectabledegradation.

Example 11 Recombinant RSV F Micelle Activity in Cotton Rats

In this example, animals groups included immunization at days 0 and 21with live RSV (RSV), formalin inactivated RSV (FI-RSV), RSV-F protein BV#683 with and without aluminum (PFP and PFP+Aluminum Adjuvant), and PBScontrols.

As shown in FIG. 21 , immunization with 30 ug of the F.-micelle vaccine(RSV-F protein BV #683, i.e. F protein 683, SEQ ID NO: 8), with andwithout aluminum produced robust neutralizing antibody responsesfollowing exposure to both RSV A and RSV B. In addition, it was observedthat aluminum significantly enhances the antibody response. Moreover,neutralizing antibodies were increased following a boost at day 46 orday 49 in RSV A and RSV B, respectively.

While significant lung pathology was observed in rats immunized withformalin inactivated RSV (FI-RSV), no disease enhancement was seen withthe F-micelle vaccine (FIG. 22 ). The use of the F-micelle vaccine andthe F-micelle vaccine with adjuvant produced lower inflammation scores(4.0 and 2.8, respectively) than the primary RSV infection (PBS+RSVchallenge) control group (5.8). As noted above, the FI-RSV treated grouphad a higher inflammation score than the primary RSV infection (PBS+RSVchallenge) control group (9.0 versus 5.8). Moreover, the FI-RSV treatedgroup had a significantly higher mean inflammation score (9.0) than theunchallenged placebo controls, live RSV+RSV challenge, F-micelle+RSVchallenge, and F-micelle aluminum+RSV challenge.

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodtherefrom as modifications will be obvious to those skilled in the art.It is not an admission that any of the information provided herein isprior art or relevant to the presently claimed inventions, or that anypublication specifically or implicitly referenced is prior art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Although the application has been broken into sections to direct thereader's attention to specific embodiments, such sections should be notbe construed as a division amongst embodiments. The teachings of eachsection and the embodiments described therein are applicable to othersections.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1. (canceled)
 2. An immunogenic formulation comprising an RSV Fglycoprotein comprising: (i) an inactivated primary furin cleavage site;wherein the primary furin cleavage site is inactivated by amino acidsubstitution of one or more amino acids of arginine 133, arginine 135,and arginine 136; (ii) a wild-type secondary cleavage site having anamino acid sequence of RARR SEQ ID NO: 23), (iii) a p27 protein; (iv) atransmembrane domain; and (v) up to four additional mutations; whereinthe RSV F glycoprotein lacks an N-terminal signal peptide; wherein theRSV F glycoprotein is cleaved into F1 and F2 subunits which aredisulfide linked; wherein the p27 protein is connected to the F1 subunitvia an amide bond; and wherein the amino acids of the RSV F glycoproteinare numbered according to the RSV glycoprotein of SEQ ID NO:
 2. 3. Theimmunogenic formulation of claim 2, wherein one amino acid of arginine133, arginine 135, and arginine 136 is substituted.
 4. The immunogenicformulation of claim 2, wherein two amino acids of arginine 133,arginine 135, and arginine 136 are substituted.
 5. The immunogenicformulation of claim 2, wherein each of arginine 133, arginine 135, andarginine 136 are substituted.
 6. The immunogenic formulation of claim 3,wherein the one amino acid is substituted with glutamine.
 7. Theimmunogenic formulation of claim 4, wherein the two amino acids aresubstituted with glutamine.
 8. The immunogenic formulation of claim 5,wherein each of arginine 133, arginine 135, and arginine 136 aresubstituted with glutamine.
 9. The immunogenic formulation of claim 2,wherein the RSV F glycoprotein comprises a deletion of up to 10 aminoacids of the fusion domain corresponding to amino acids 137-146 of SEQID NO: 2,
 10. The immunogenic formulation of claim 2, wherein the RSV Fglycoprotein comprises a deletion of the fusion domain corresponding toamino acids 137-146 of SEQ ID NO:
 2. 11. The immunogenic formulation ofclaim 8, wherein the RSV F glycoprotein comprises a deletion of thefusion domain corresponding to amino acids 137-146 of SEQ ID NO:
 2. 12.The immunogenic formulation of claim 2, comprising an adjuvant.
 13. Theimmunogenic formulation of claim 2, wherein the RSV F glycoproteincomprises two additional mutations.
 14. The immunogenic formulation ofclaim 2, wherein the RSV F glycoprotein comprises four additionalmutations.
 15. The immunogenic formulation of claim 11, wherein the RSVF glycoprotein comprises two additional mutations.
 16. The immunogenicformulation of claim 11, wherein the RSV F glycoprotein comprises fouradditional mutations.
 17. A method of eliciting an immune response to anRSV infection in a human comprising administering the immunogenicformulation of claim
 2. 18. The method of claim 17, wherein the human isan infant.
 19. The method of claim 17, wherein the administering isintramuscular.